Induction of Intrinsic and Extrinsic Apoptosis Pathways in the Human Leukemic MOLT-4 Cell Line by Terpinen-4-ol

Apoptosis is a process by which cells undergo organized self-destruction without an inflammatory response. Apoptotic cell morphologies include membrane blebbing, nuclear and cytoplasmic condensation and apoptotic bodies (Schimmer et al., 2001). In the extrinsic pathway, ligation of the death receptors stimulates the activation of the initiator caspase-8, which then triggers downstream events either by directly activating caspase-3 or Bid cleavage. Truncated-Bid in turn initiates the mitochondrial pathway. Pro-apoptotic Bid targets the mitochondria and regulates other apoptotic-related protein such as Bcl-2, Bcl-xL and Bax (Reyes-Zurita et al., 2011). The intrinsic pathway plays a key role in regulating cell death through mitochondria. Mitochondrial outer membrane permeabilization (MOMP) decides the ‘‘point of no return’’ of apoptotic cell death and triggers the release into the cytoplasm of proteins that mediate cell death, such as cytochrome c. This leads to the activation of caspase-9, which then triggers effector caspase-3 activation, resulting in apoptotic cell death (Kang and Reynolds, 2009). Terpinen-4-ol is the main component from rhizomes of “Plai” in Thai (Zingiber montanum (Koenig) Link ex Dietr.) (Bua-in and Paisooksantivatana, 2009). It is used as herbal medicine with several activities, such as antiinflammation (Hart et al., 2000), anti-hypersensitivity


Introduction
Apoptosis is a process by which cells undergo organized self-destruction without an inflammatory response. Apoptotic cell morphologies include membrane blebbing, nuclear and cytoplasmic condensation and apoptotic bodies (Schimmer et al., 2001). In the extrinsic pathway, ligation of the death receptors stimulates the activation of the initiator caspase-8, which then triggers downstream events either by directly activating caspase-3 or Bid cleavage. Truncated-Bid in turn initiates the mitochondrial pathway. Pro-apoptotic Bid targets the mitochondria and regulates other apoptotic-related protein such as Bcl-2, Bcl-xL and Bax (Reyes-Zurita et al., 2011). The intrinsic pathway plays a key role in regulating cell death through mitochondria. Mitochondrial outer membrane permeabilization (MOMP) decides the ''point of no return'' of apoptotic cell death and triggers the release into the cytoplasm of proteins that mediate cell death, such as cytochrome c. This leads to the activation of caspase-9, which then triggers effector caspase-3 activation, resulting in apoptotic cell death (Kang and Reynolds, 2009).

Induction of Intrinsic and Extrinsic Apoptosis Pathways in the Human Leukemic MOLT-4 Cell Line by Terpinen-4-ol
Patompong Khaw-on, Ratana Banjerdpongchai* (Lahlou et al., 2003), anti-bacterial (Loughlin et al., 2008) and anti-fungal infection (Mondello et al., 2006). Furthermore, it can impair the growth of human M14 melanoma cells (Calcabrini et al., 2004) as well as induces necrotic cell death coupled with apoptotic cell death and arrests cell cycle at G1 stage in mouse malignant mesothelioma AE17 and murine melanoma B16-F10 cells (Greay et al., 2010). Further studies of potential anti-cancer drugs with a variety of cell lines are necessary, because there are high variability responses in different cancer cells following treatment with cytotoxic agents. The aims of this study were to investigate mode of cell death and the mechanisms of terpinen-4-ol-induced cytotoxicity in human leukemic MOLT-4 cell line.
Mouse monoclonal antibodies to cytochrome c and betaactin were obtained from Santa Cruz Biotechnology, USA. Annexin-V-FLUOS staining kit was obtained from Roche, Basel, Switzerland. BCA protein assay kit was obtained from Bio-rad, Hercules, CA, USA. SuperSignal West Pico Chemiluminescent Substrate was purchased from Pierce, Rockford, IL, USA.

Cell culture and Cytotoxicity test
Human acute lymphoblastic leukemia MOLT-4 cells were gifts from Dr. Watchara Kasinroek (Faculty of Associated Medical Science, Chiang Mai University, Thailand). Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood by density gradient centrifugation using the standard protocols. The cells were cultured in 10% fetal bovine serum in RPMI-1640 medium supplemented with penicillin G (100 units/ml) and streptomycin (100 μg/ml) at 37°C in a humidified atmosphere containing 5% CO 2 . MOLT-4 cells were grown in the presence of terpinen-4-ol at concentrations ranging from 10 to 160 μM. Following terpinen-4-ol treatment, cell viability was assessed by MTT (3-(4, 5-dimethyl)-2, 5-diphenyl tetrazolium bromide) assay. Briefly, MTT dye solution was added and incubated in CO 2 incubator for 4 h. Then, 100 μl of DMSO was added to dissolve the dye crystals. Absorbance was measured by using a microplate reader (Biotek, USA) at 570 nm. The percentage of cell viability was calculated and 20% and 50% inhibitory concentrations (IC 20 and IC 50 ) were determined.

Determination of cell cycle distribution and Apoptosis assay
The effect of terpinen-4-ol on MOLT-4 cell cycle phase distribution was assessed using flow cytometry. Briefly, after cell treatment with terpinen-4-ol for various times, cells were harvested and thereafter washed twice with cold PBS, and centrifuged. The pellet was resuspended in 500 μl cold 70% methanol in PBS for overnight at 4°C. The cells were centrifuged at 750 x g for 5 min, pellets were washed twice with cold PBS, suspended in 500 μl PBS, incubated with 10 μl RNAase (20 μg/ml final concentration) and stained with PI (50 μg/ml final concentration) overnight at 4°C before analysis by flow cytometry. DNA histograms were further analyzed using FACS DIVA software (Becton Dickinson, USA) for cell cycle analysis. Percentage of cells in each phase was evaluated.
Apoptosis was determined with the annexin-V-FLUOS staining kit according to the manufacturer's instructions. To examine apoptotic cell morphology by using the Wright-Giemsa staining method, cells were stained with Wright-Giemsa dye and observed under light microscope (Jia et al., 2003).

Reduction of mitochondrial transmembrane potential (ΔΨm)
Reduction of mitochondrial transmembrane potential (ΔΨm) during the induction of apoptosis was examined by using 3,3-dihexyloxacarbocyanine iodide (DiOC 6 ) (Zamzami et al., 2007). Cells were harvested after terpinen-4-ol treatment, and DiOC 6 was added to final concentration of 40 nM. After 15 minutes of incubation at 37°C, the cells were washed and analyzed by flow cytometry technique.

Western blot analysis
After treatment, cells were washed twice with PBS and lysed in 100 μl of RIPA buffer at 4°C for 15 minutes, then centrifuged at 12,000 x g for 15 minutes. The precipitates were removed and the solutions stored at -80°C. Concentration of protein was determined by the BCA protein assay kit using bovine serum albumin as a standard. Western blot analysis was conducted by a method described previously (Banjerdpongchai et al., 2010). Briefly, cellular protein 50 μg was loaded onto 15% SDS-polyacrylamide gels. The protein bands were transferred to nitrocellulose membranes and probed with anti-cytochrome c, anti-Bcl-2 and anti-truncated Bid, followed by a horseradish peroxidase-conjugated secondary antibody. Detection of the antibody reactions was performed by enhanced chemiluminescent reagents and chemiluminescence was exposed to the X-ray films. The bands were analyzed by densitometer.

Statistical analysis
Data were expressed as mean±S.D. (standard deviation). Statistical difference between control and treated group was determined by the one-way ANOVA (Kruskal Wallis analysis) in triplicate of three independent experiments. Statistical significance is expressed as *p <0.05, **p <0.01.

Cell cytotoxicity
Terpinen-4-ol was cytotoxic to MOLT-4 cells in a dose dependent manner but not affect PBMCs as shown in Figure 1. The IC 50 and IC 20 (inhibitory concentration at 50% and 20%) values of terpinen-4-ol on MOLT-4 cells are 155 and 45 μM, respectively. Then the inhibitory concentration of terpinen-4-ol at 50% (IC 50 ) or 20% (IC 20 ) was used for further experiments.

Effect of terpinen-4-ol on the apoptosis induction
To examine whether terpinen-4-ol could induce apoptosis in MOLT-4 cells, the induction of apoptosis by terpinen-4-ol was examined for the morphology. The appearance of nuclear condensation and apoptotic bodies was demonstrated, which are characteristic of apoptotic cells by using Wright-Giemsa staining (Figure 2A). The increase in annexin-V-positive cell populations was found ( Figure 2B). In addition, Sub-G1 DNA contents were Induction of      measured by cell cycle analysis ( Figure 2C). MOLT-4 cells treated with terpinen-4-ol exhibited time-dependent increase in hypodiploid (<2n DNA) population. Terpinen-4-ol significantly increased the percent accumulation of sub-G1 populations from control (8.70%) to 11.00, 11.83 and 17.47% at 0, 6, 12, and 24 h of treatment, respectively ( Figure 2D).

ΔΨm loss and release of cytochrome c from mitochondria of MOLT-4 cells treated with terpinen-4-ol
To study the mechanisms of apoptosis involved in terpinen-4-ol-induced MOLT-4 cell apoptosis, mitochondrial transmembrane potential and cytochrome c release were investigated by flow cytometry analysis and immunoblotting. In the untreated control cells, almost all cells were functionally active with high DiOC 6 fluorescence; percentage of cells with ΔΨm was low. Terpinen-4-ol at 40 μM caused mitochondrial damage and percent cells with the decrease of mitochondrial transmembrane potential was about 50% ( Figure 3A). The loss of MTP is largely due to the opening of mitochondrial permeability transition pores (PTP), which conducted the leakage of cytochrome c from mitochondria to the cytosol at 40 μM ( Figure 3B). These results suggest a role of the mitochondria in terpinen-4-ol-induced apoptosis.
Bcl-2 family proteins including proteins that suppress apoptosis, such as Bcl-2 and proteins that promote apoptosis, such as Bad, Bax, and Bid, have been reported to play a central role in regulating cytochrome c release from mitochondria (Brunelle and Letai, 2009). Terpinen-4-ol decreased Bcl-2 expression at 24 h treatment by immunoblotting ( Figure 4B). Moreover, proapoptotic t-Bid was elevated at 24 hours after terpinen-4-ol exposure. Taken together, altered expression of Bcl-2 was involved with the presence of truncated-Bid as a connection between the extrinsic and the intrinsic apoptotic pathways.
Terpinen-4-ol contains antiproliferative and antitumor effects in human nonsmall lung cancer cells (Wu et al., 2012). The present study revealed that terpinen-4-ol induced apoptosis in human leukemic cells in vitro via mitochondria-mediated intrinsic pathway and caspase-8activated extrinsic pathways.