Alpha-phellandrene induced apoptosis in mice leukemia WEHI-3 cells
in vitro
Jen-Jyh Lin1,2, Shu-Chun Hsu3, Kung-Wen Lu1, Yi-Shih Ma4,5, Chih-Chung Wu6, Hsu-Feng Lu7, Jaw-Chyun Chen8, Jaung-Geng Lin1, Ping-Ping Wu9,*, and Jing-Gung
Chung3,10,*
1Graduate Institute of Chinese Medicine, China Medical University, Taichung 404, Taiwan; 2Division of Cardiology, China Medical University Hospital, Taichung 404, Taiwan; 3Department of Biological Science and Technology, China Medical University, Taichung 404, Taiwan; 4School of Chinese Medicine for Post-Baccalaureate, I-Shou University, Kaohsiung 84001, Taiwan; 5Department of Chinese Medicine, E-Da Hospital, Kaohsiung 82445, Taiwan; 6Department of Nutrition and Health Science, Chang Jung Christian University, Tainan 711, Taiwan; 7Department of Clinical Pathology, Cheng Hsin General Hospital, Taipei, 112, Taiwan; 8Department of Medicinal Botany and Health Applications, Da-Yeh University, Changhua 51591, Taiwan; 9School of Pharmacy, China Medical University, Taichung 404, Taiwan; 10Department of Biotechnology, Asia University, Taichung 413, Taiwan, R.O.C.
Running title: α-phellandrene induced apoptosis in mice leukemia WEHI-3 cells.
*Both have equal contribution
*Address correspondence to Prof. Jing-Gung Chung, Department of Biological Science and Technology, China Medical University, No 91, Hsueh-Shih Road, Taichung 404, Taiwan. Tel: +886 4 2205 3366 ext 2531, Fax: +886 4 2205 3764, e-mail: [email protected]
Ping-Ping Wu, School of Pharmacy, China Medical University, No 91, Hsueh-Shih Road, Taichung 40402, Taiwan. Tel: +886-4-22053366 ext 5108, Fax: +886-4-22031075, e-mail: ppwu @mail.cmu.edu.tw
Abstract. Although reports have shown that α-phellandrene (α-PA) is one of the
monoterpenes and is often used in the food and perfume industry, our previous studies have indicated that α-PA promoted immune responses in normal mice in vivo. However, there is no available information to show that α-PA induced cell apoptosis in cancer cells, thus, we investigated the effects of α-PA on the cell morphology, viability, cell cycle distribution and apoptosis in mice leukemia WEHI-3 cells in vitro. Results indicated that α-PA induced cell morphological changes and decreased viability, induced G0/G1 arrest and sub-G1 phase (apoptosis) in WEHI-3 cells. α-PA increased the productions of reactive oxygen species (ROS) and Ca2+ and decreased the levels of mitochondrial
membrane potential (ΔΨm) in dose- and time- dependent manners in WEHI-3 cells that
were analyzed by flow cytometer. Results from confocal laser microscopic systems examinations show that α-PA promoted the releases of cytochrome c, AIF and Endo G from mitochondria in WEHI-3 cells. These results are the first findings to provide new information for understanding the mechanisms by which α-PA induces cell cycle arrest and apoptosis in WEHI-3 cells in vitro.
Keywords: α-phellandrene (α-PA); WEHI-3 cells; cell cycle arrest; apoptosis; in vitro
Introduction
Leukemia, a myeloproliferative disease that was characterized via the abnormal growth of phenotypically immature leukocytes, is one of the common causes of death in human populations. Moreover, the incidence of this cancer is increasing worldwide. Based on 2010 reports from the “Bureau of National Health Insurance”, it was demonstrated that about 4.2 persons per 100 thousand people die per year from leukemia in Taiwan. However, the treatments of leukemia including chemotherapy, radiotherapy, and combined with chemotherapy, radiotherapy are still not satisfactory, though they do improve survival rates of patients with these diseases . Thus, therapies with increased efficacy and decreased toxicity are needed to treat leukemia patients.
It was reported that modifications of life style and diets can prevent cancer development in human and consumption of plant-based diet can decrease colon cancer occurrence . Thus, investigators were focused on the findings of natural agents from plant
for treating cancer in human. Schinus molle L have been shown to have biological functions such as antiseptic and fungi toxicant and anti-tumoral as well as antispasmodic and analgesic . The extract of leaf from Schinus molle L had been shown to have antidepressant , analgesic and central depressant . The α-PA is a major component of
Schinus molle L essential oil (>50%) and it was reported in the plant Aegle marmelos
(L.). Correa is a sacred medicinal and nutraceutical tree of India . The α-PA is one of the monoterpenes and it was often used in the food and perfume industry .
However, no previous studies on the pharmacological and toxicological effects of α-PA were reported in the literature. The aim of this study was to evaluate the effects of cytotoxity of α-PA on human leukemic cells in vitro. The results indicated that α-PA induced apoptosis in WEHI-3 cells through the induction of death receptor, mitochondrial and endoplasmic reticulum stress pathways.
Materials and Methods
Chemicals and reagents. α-phellandrene (α-PA) was kindly offered by Dr. Wu
(Department of Nutrition and Health Science, Chang Jung Christian University, Tainan, Taiwan). Dimethyl sulfoxide (DMSO), potassium phosphates, propidium iodide, ribonuclease-A, Tris-HCl Trypan blue and Triton X-100 were obtained from Sigma Chemical Co. (St. Louis, MO, USA). 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA, Sigma) for ROS measurement, DiOC6 for the measurement of mitochondria membrane potential (ΔΨm) and Indo 1/AM for Ca2+ measurements were purchased from
Calbiochem (Darmstadt, Germany). RPMI-1640 medium, glutamine, fetal bovine serum (FBS) and penicillin-streptomycin, trypsin-EDTA were purchased from Invitrogen (Carlsbad, CA, USA).
Cell culture. The WEHI-3 cell line was purchased from the Food Industry Research and
Development Institute (Hsinchu, Taiwan). The cells were grown in RPMI-1640 medium containing 10% (v/v) fetal bovine serum (FBS), 1% penicillin-streptomycin (100 U/ml penicillin and 10 g/ml streptomycin) and 1% glutamine in a 37°C humidified incubator with 5% CO2. The cells were subcultured at 80-90% confluency .
Observation of morphological changes and assessment of cell viability. WEHI-3 cells
were seeded into 24-well plates at the density of 2x105 cells/well in 1 ml medium and incubated at 37°C for 24 h before each well were individually treated with 0, 5, 10, 30, 40 and 50 μM of α-PA for 24 and 48 h. For cell morphological changes determination, the cells were observed under a phase-contrast microscope and photographed . For cell viability determination, at the end of treatment, cells were harvested from above each treatment and then were analyzed for the percentage of viable cells by flow cytometric protocol as previously described .
Cell cycle analysis. To examine the cell cycle distribution, WEHI-3 cells (2x105 cells/well) in 24-well plate were incubated with or without α-PA (0, 5, 10, 30, 40 and 50 μM) for 24 h before cells were fixed in ethanol. The fixed cells were washed with PBS and then were stained by PI for 5 min and analyzed by flow cytometer (BD Bioscience, San Diego, CA, USA). The percentage of cells in the sub-G1 (apoptosis), G0/G1-, S- and G2/M-phases were analyzed by using ModFit LT 3.0 program as described previously mentioned .
Flow cytometery assay for apoptosis using Annexin V/PI staining. To examine the
percentage of apoptosis, WEHI-3 cells (2x105 cells/well) in 24-well plate were incubated with or without α-PA (0 and 10 μM) for 0, 1, 3, and 6 h, and the cells were harvested and stained with Annexin V/PI staining Kit according to the manufacturer’s instructions and as we previously described . Then the percentages of earlier and late apoptotic cells were calculated as previously .
Detection of reactive oxygen species (ROS), Ca+2 production and levels of mitochondrial membrane potential. WEHI-3 cells (2x105cells/well) in 24-well plate were treated with 10 μM of α-PA for 0, 1, 3, 6, 12 and 24 h. The cells from each treatment were harvested and were washed twice by PBS before being re-suspended in 500 μl of 2,7-dichlorodihydrofluorescein diacetate (10 μM) (DCFH-DA, Sigma) for ROS measurements, in 500 μl of Indo 1/AM (3 μg/ml) (dye contains fluorescence for staining
of Ca2+) and in 500 μl of DiOC6 (5 μg/ml) (dye contains fluorescence for staining of
ΔΨm ) (Darmstadt, Germany). All samples were incubated at 37oC for 30 min to detect percentage of changes in ROS, Ca2+ and MMP by using flow cytometry, as described previously (Becton Dickinson FACS Calibur) .
Detection of caspase-8, -9 and -3 activities. WEHI-3 cells (2x105 cells/well) in 24-well plate were treated with 10 μM of α-PA for 0, 6, 12 and 24 h. All samples were harvested and washed twice with PBS before adding caspase-8, -9 and -3 substrates (CaspaLux 8-L1D2,- CaspaLux 9-M1D2 and PhiPhiLux-G1D2), respectively, then the activities of caspase-8, -9 and -3 were determined by using flow cytometric assay as described previously .
Confocal laser scanning microscopy for protein translocation. WEHI-3 cells (5×104 cells/well) were maintained on 4-well chamber slides and then were treated without or with 10 μM of α-PA for 24 h. Cells were fixed in 4% formaldehyde in PBS for 15 min, permeabilized with 0.3% Triton-X 100 in PBS for 1 h with the blocking of non-specific binding sites using 2% BSA . All control and α-PA-treated fixed cells were separately stained with anti-cytochrome c, anti-AIF and anti-Endo-G (1:100 dilution) (green fluorescence) as primary antibodies overnight. All samples were washed twice with PBS and then were stained with FITC-conjugated goat anti-mouse IgG at 1:100 dilution as secondary antibody, and then were further stained with PI (red fluorescence) for DNA staining. All samples were examined and photomicrographed by using a Leica TCS SP2 Confocal Spectral Microscope .
Western blot analysis. WEHI-3 cells at a density of 1x107 cells in 75 T flasks were incubated with 10 µM α-PA for 0, 6, 12, 24 and 48 h for examining the protein levels correlated with apoptosis. At the end of incubation, cells from each treatment were collected, and the total protein lysates was isolated, gel electrophoresis and immunoblotting were conducted as previously described . The primary antibodies were anti-Fas, Fas-L, Caspase-8, AIF, Endo G, Cytochrome c, Caspase-9, Caspase-3, PARP, Bax, Bcl-2, Bad, tBid, Mcl-1, ATF-6α, GRP78, GADD153, Caspase-12 and β-actin.
Immunoreactive proteins of all examined samples were visualized with the ECL chemiluminescent detection system (Perkin-Elmer Life Science, MA, USA) and BioMax Light Film (Eastman Kodak, New Heaven, CT, USA) according to the manufacturer's instructions .
Statistical analysis. All data were expressed as mean±S.D. from three individual
experiments. Statistical calculations of the data were performed using an unpaired Student’s t-test. p values <0.05 were considered statistically.
Results and discussion
α-PA induced cell morphological changes and decreased the percentage of viable cells in WEHI-3 cells. WEHI-3 cells were treated with various concentrations of α-PA for various
time periods before cells were examined and photographed for cell morphological changes and the results are shown in Figure 1A. The pictures from figure 1A indicated that the higher α-PA concentration led to a lower amount of viable cells and also more damaged cells when compared to the control groups and these effects are in a dose-dependent response. For total viable cell determination, cells were used by propidium iodine staining then analyzed by flow cytometry and the results are shown in Figure 1B, which indicated that there were fewer viable cells as time and concentration of α-PA increased when compared to control groups and these effects are dose- and time-dependent manners (p<0.05). The cytotoxic effects of α-PA on WEHI-3 cells were observed.
α-PA induced G0/G1 arrest and sub-G1 in WEHI-3 cells. WEHI-3 cells were treated with
various concentrations of α-PA for 24 h before cells were harvested and stained with PI and then were analyzed by flow cytometry for cell cycle distribution and sub-G1 phase and the results are shown in Figure 2A and B. As shown in Figure 2A and B, there was an increase in the percentage of cells in G0/G1 and a decrease in the percentage of cells in S and G2/M phases. The sub-G1 phase appeared in the cell cycle distribution, suggesting that α-PA induced apoptosis in WEHI-3 cells (Fig. 2B). Increased doses of time of α-PA led to increase the percentage of cells in sub-G1 phase (p<0.05).
α-PA induced apoptosis in WEHI-3 cells. Figure 2A already showed that α-PA induced
sub-G1 phase occurrence. Thus, we further confirm whether α-PA induced apoptosis in earlier treated time. WEHI-3 cells were treated with 10 μM of α-PA for 0, 1, 3 and 6 h before cells were harvested and stained with Annexin V/PI and then were analyzed for apoptosis and the results are shown in Figure 3. The results indicated that α-PA induced apoptosis in the examined time periods (1-6 h) and these effects are time-dependent.
α-PA affected the production of reactive oxygen species (ROS) and space Ca2+and the levels of mitochondria membrane potential (ΔΨm) in WEHI-3 cells. WEHI-3 cells were
treated with 10 μM of α-PA for various time-periods, before all samples were harvested for ROS and Ca2+productions, loss of mitochondrial ΔΨm analyzed and quantified and the results are shown in Figure 4A, B and C. These figures showed that α-PA induced ROS production quite early (1h treatment) (Fig. 4A). Figure 4B indicated that α-PA promoted Ca2+ productions in WEHI-3 cells at 24 h treatment. Figure 4C showed that α-PA decreasedthe levels of ΔΨm in a time-dependent manner.
α-PA induced caspase-8, -9 and -3 activities in WEHI-3 cells. In order to investigate
whether caspases are involved in α-PA induced apoptosis, WEHI-3 cells were treated with α-PA (10 μM) for various time before cells were harvested and activities of caspase-8, -9 and -3 were determined and the results are shown in Figure 5A, B and C. These figures indicated that 10 μM of α-PA promoted caspase-8 (Fig. 5A), -9 (Fig. 5B) and -3 (Fig. 5C) activities in a time-dependent manner. These findings showed that caspase-8, -9 and -3 are involved in α-PA induced apoptosis in WEHI-3 cells.
α-PA affected Cytochrome c, AIF and Endo-G distribution in WEHI-3 cells. In order to
further investigate whether apoptotic protein migration from cytoplasm to nuclei are involved in α-PA induced apoptosis in WEHI-3 cells, WEHI-3 cells were treated with or without α-PA (10 μM) for 24 h and then were examined and photographed by confocal laser microscope and the results are shown in Figure 6A, B and C. These figures indicated that cytochrome c (Fig. 6A) was released from mitochondria compared to
control sample which have lower green color. AIF (Fig. 6B) and Endo-G (Fig. 6C) are also released from mitochondria when compared to the control group. These results also indicated that α-PA induced apoptosis in WEHI-3 cells are of a mitochondria dependent manner. Results from confocal laser microscopy examination (Fig. 6A, B and C) indicated that α-PA promoted the releases of cytochrome c, AIF and Endo G from mitochondria. Based on these observations, we suggest that α-PA induced apoptosis in WEHI-3 cells through mitochondria-dependent pathways.
α-PA alters levels of proteins associated with apoptosis in WEHI-3 cells. For
investigating the possible signaling pathways of α-PA induced apoptosis in WEHI-3 cells, the cells were treated with 10 M of α-PA for 0, 6, 12, 24 and 48 h before analysis of the protein level change by Western blotting. The results are shown in Figures 7A, B, C and D. α-PA increased the expressions of Fas, Fas-L, Caspase-8 (Fig.6A), Bax, Bad, tBid (Fig. 6B), AIF, Endo G, cytochrome c, Caspase-9, Caspase-3, PARP (Fig. 6C), ATF-6α, GRP78, GADD153 and Caspase-12 (Fig. 6D) but decreased the expressions of Bcl-2 and Mcl-1 (Fig. 6B) in WEHI-3 cells. Therefore, we suggesting that α-PA induced apoptosis in WEHI-3 cells may through the induction of death receptor, mitochondrial and endoplasmic reticulum stress pathways.
Conclusion
In our primary studies we have shown that α-PA promoted immune responses including increased macrophage phagocytosis and natural killer cells activity in animal model in vivo . However, there is no any report to show α-PA induced apoptosis in mice leukemia cell lines. Thus, in the present study, we investigated the cytotoxic effects of α-PA in mice leukemia WEHI-3 cells in vitro. Herein, our results demonstrated that α-α-PA induced morphological changes and decreased the viable cells (Fig. 1A and B), and induced G0/G1 arrest and sub-G1 phase (Fig. 2A and B) in WEHI-3 cells. These effects are doses-dependent manners.
It is well known that agents induced sub-G1 phase in cell cycle distribution of cancer cells which means that this agent can induce apoptosis . Thus we further investigated the apoptotic cells by using Annexin V/PI staining and then analyzed by using flow
cytometry methods and the results are shown in Figure 3 which indicated that α-PA induced apoptosis in earlier treated time start at 1h to 6h treatment. The Annexin V/PI staining for examining the cell apoptosis are well established .
It is well documented that apoptosis can be divided into the mitochondria-independent and -dependent pathways , former pathway via caspase-8 then directly to activate caspase-3 for causing apoptosis but the later pathways involved the dysfunction of mitochondria before leading to cytochrome c, AIF and Endo G release from mitochondria and led to apoptosis. Results from Figure 4A, B and C indicated that α-PA promoted the production of ROS and Ca2+ but decreased the levels of ΔΨm in WEHI-3 cells. Ca2+ release also showed the ROS production and mitochondria dysfunction which led to cytochrome c, AIF and Endo G release from mitochondria.
It is also well known that agents induced apoptosis via caspase-dependent and -independent pathways , herein, our results from Figure 5A, B and C indicated that α-PA promoted the activities of caspase-8, -9 and -3. Based on these observations, we suggested that α-PA induced apoptosis in WEHI-3 cells through the caspase-dependent pathway.
In conclusion, the present results demonstrated the α-PA induced cytotoxic effects of WEHI-3 cells via the induction of cell apoptosis. Moreover α-PA promoted ROS and Ca2+ productions, decreased the levels of ΔΨm for cytochrome c, AIF and Endo G release from mitochondria followed by the activations of caspase- 9 and -3 and finally led to apoptosis in WEHI-3 cells in vitro that is summarized in Figure 8. Taken together, these findings provide new insights into the possible pathway and function in vitro of α-PA in mice WEHI-3 cells.
Acknowledgement
This work was supported by grant DOH102-TD-C-111-005 from Department of Health, Executive Yuan, R.O.C (Taiwan). Experiments and data analysis were performed in part through the use of the Medical Research Core Facilities Center, Office of Research & Development at China medical University, Taichung, Taiwan, R.O.C.
The authors declare that there are no conflicts of interest.
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Figure legends
Figure 1. α-PA induced cell morphological changes and the percentage of viable cells in
WEHI-3 cells. Cells were placed in RPMI-1640 + 10% FBS with 0, 5, 10, 30, 40 and 50 μM of α-PA for 24 h and then were examined and photographed under contrast phase microscope (A) or were treated with α-PA (0, 5, 10, 30, 40 and 50 μM) for 24 and 48 h (B) for percentages of viable cells.
Figure 2. α-PA induced G0/G1 arrest and sub-G1 in WEHI-3 cells. Cells were cultured
in RPMI-1640 + 10% FBS with various concentrations of α-PA for 24 h for cell cycle distribution assay. The cells were analyzed for cell cycle distribution (A) and quantitated in various phases of cell cycle (B) by flow cytometry as described in Materials and Methods. Each point is mean ± S.D. of three experiments. *p<0.05; ***p<0.001
Figure 3. α-PA induced apoptosis was examined by using Annexin V/PI staining in
WEHI-3 cells. To confirm cell apoptosis, WEHI-3 cells (2x105cells/well) in 24-well plate were treated with 10 μM of α-PA for 0, 1, 3 and 6 h before then cells from each well were harvested for staining of annexin V/PI, then analyzed by flow cytometry assay as described in Materials and Methods.
Figure 4. α-PA affected the productions of reactive oxygen species (ROS) and Ca2+ and the levels of mitochondria membrane potential (ΔΨm) in WEHI-3 cells.Cells were treated
with 10 μM of α-PA for 0, 1, 3, 6, 12 and 24 h before being stained by 2,7-dichlorodihydrofluorescein diacetate for ROS levels determined (A), by Indo 1/AM for Ca2+ levels determined (B) and by DiOC6 for the ΔΨ
in Materials and Methods. Data represents mean±S.D. of three experiments. *p<0.05.
Figure 5. α-PA induced caspase-8, -9 and -3 activity in WEHI-3 cells. Cells were treated with 10 μM of α-PA for various time periods, and cells were harvested for measuring the activities of caspase-8 (A), caspase -9 (B) and caspase-3 (C) as described in Materials and Methods. Data represents mean±S.D. of three experiments. *p<0.05.
Figure 6. α-PA promoted the levels of cytochrome c, AIF and Endo-G in WEHI-3 cells.
Cells were incubated with 10 μM of α-PA for 24 h and then were fixed and stained with anti-cytochrome c (A), anti-AIF (B) and anti-Endo G (C) which were then stained by FITC-labeled secondary antibodies (green fluorescence) and the nuclei were stained by PI (red fluorescence). All stained proteins were examined and photographed by a confocal laser microscopic system. Scale bar, 20 μm.
Figure 7. α-PA affect the levels of associated proteins in apoptosis of WEHI-3 cells.
Cells (5x105 cells/well) were treated with α-PA (10 μM) for different time periods and then the total protein were determined and used for SDS page gel electrophoresis as described in Materials and Methods. The levels of Fas, Fas L, Caspase-8 (A), Bax, Bcl-2, Bad, tBid, Mcl-1 (B) AIF, Endo G, cytochrome c, caspase-9, caspase-3, PARP (C), ATF-6, GRP78, GADD153, Caspase-12 (D) expressions were estimated by Western blotting as described in Materials and Methods.
