Ⅰ.INTRODUCTION
Cell death is traditionally classified into 2 types; apoptosis and necrosis forms1,2). Apoptosis is considered programmed cell death, which is controlled by elaborated coordinated pathway3). Meanwhile, necrosis is well known as non-programmed cell death, which occurs in response to external shocks to the cells or tissue, such as trauma, infection, or toxin, which can result in the unregulated digestion of cell components. It is characterized by morphological features including the increase of cell volume, microorganelle swelling, the rupture of plasma membrane and subsequent cell death4).
Recently, a new type of cell death, necroptosis, has been reported. Numerous reports identified necroptosis as a programmed cell death mechanism, not cell death caused by accidental chance. Necroptosis can be initiated when apoptosis is shut out. In addition, apoptosis is associated with caspase-dependent pathway while necroptosis is associated with kinase, especially receptor interacting protein (RIP), a sort of kinase activity containing protein, which is essential in the detection of cellular stress 5,6). Recently, multiple lines of evidence have demonstrated that necroptosis is associated with myocardial infarction7), cerebral infarction 8,9), atherosclerosis10), acute pancreatitis 11), and more importantly, cancer 12,13).
The mature fruit of Kochia scoparia (L.) Schrad. (family Chenopodiaceae) is widely spread throughout the western and northern United States, Europe, Africa, South America, and the Far East14). The plant has been used in Chinese and Korean traditional medicine as a treatment for allergic skin diseases15), diabetes mellitus16), rheumatoid arthiritis17) and liver diseases18). In addition, the fruit is used as a food garnish in Japan19). Several recent studies indicated that the extract of K. scoparia fruit has anti-tumor activity against human hepatocellular carcinoma cells and immortal neuroblastoma cell lines20-22). Previously, the authors reported that the methanol extract of K. scoparia dried fruit (MEKS) has possible anti-cancer effects in human breast cancer cell lines and oral squamous cell carcinoma cell (OSCC) lines. The main mechanism of MEKS was reported to be apoptosis, which was controlled by the activation of caspases. However, no report has been issued on the other anti-cancer mechanism of K. scoparia in OSCC cell lines.
Therefore, we evaluated the anti-cancer activity of MEKS by measuring proliferation rates and non-apoptotic cell death in HSC4 human oral cancer cells. In addition, we sought to identify the molecular mechanism responsible.
Ⅱ.MATERIALS AND METHODS
Reagents and antibodies
Paclitaxel, MTT (3, 4, 5-dimethyl N-methylthiazol-2-yl-2, 5-d-phenyl tetrazolium bromide), 3, 4-Dihydro-5-[4-(1- piperidinyl)butoxy]-1(2H)-isoquinoline (DPQ) and propidium iodide (PI) solution were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2, 7-Dichloro-fluorescein diacetate (DCFHDA) was obtained from Eastman Kodak (Rochester, NY, USA). The ANNEXIN V-FITC apoptosis detection kit, anti-rabbit IgG antibody and anti-mouse IgG antibody were purchased from Enzo Life Sciences (Farmingdale, NY, USA). The antibodies targeting Bax, cleaved caspase 3, cleaved caspase 8, cleaved caspase 9 and cleaved Poly (ADP-ribose) polymerase (PARP) were purchased from Cell Signaling Technology (Beverly, MA, USA), while anti-beta actin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Preparation of MEKS
The dried fruit of K. scoparia was purchased from Hwalim Medicinal Herbs (Pusan, Korea). Extraction was performed using a standard extraction process, as previously described. Briefly, 100 g of the dried fruit of K. scoparia were immersed in 1 L of methanol, sonicated for 30 min and then allowed to stand for 48 h. The obtained extract was filtered through No. 20 Whatman filter paper, evaporated under reduced pressure using a vacuum evaporator (Eyela, Japan) and lyophilized using a freeze dryer (Labconco, Kansas City, MO, USA). Finally, 4.46 g of lyophilized powder was obtained (yield, 4.46%). A sample of the lyophilized powder (MEKS, Voucher No. MH2013-006) was deposited at the Division of Pharmacology, School of Korean Medicine, Pusan National University.
Cell culture
HSC4 cell (a human oral cancer cell line) was purchased from the JCRB Cell Bank (Japan) and cultured in DMEM (Hyclone Laboratories, Logan, UT, USA) supplemented with 10% FBS (Hyclone Laboratories) and 1% penicillin/ streptomycin (Invitrogen, Carlsbad, CA, USA).
MTT assay
Proliferation rates of HSC4 cells were measured using a MTT proliferation assay. Briefly, cells were seeded in 24-well plates (5x104 per well) and cultured overnight to allow attachment. They were then treated with MEKS at the concentration of 0, 12.5, 25, 50, 75 and 100 μg/ml for 4 h, respectively, and then in fresh MEKS-free media for 20 h. Control cells were treated with vehicle (dimethyl sulfoxide; DMSO) for 4 h. MTT activities were measured in triplicate for control and experimental groups. MTT solution was added to each well, and then the cells were incubated at 37 ℃ for 4 h in a 5% CO2 atmosphere. Media were then removed and the formazan crystals produced were dissolved in 100 μl DMSO. Absorbances were read at 570 nm using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).
Cell morphology
A phase contrast microscope (Olympus, Tokyo, Japan) was used to determine the cell morphologies of untreated and MEKS-treated HSC4 cells.
ANNEXIN V and 7-AAD double staining
Cells were seeded in 6-well plates (3x105 per well), incubated overnight and treated with MEKS in indicated concentrations for 4 h. MEKS was then removed and cells were cultured in fresh media for a further 20 h. Control cells were treated with vehicle (DMSO) for 4 h. Thirty nM of paclitaxel was used as positive control. After incubation, cells were trypsinized, harvested, washed with phosphatebuffered saline (PBS), resuspended in 500 μl of Binding Buffer (ANNEXIN V-FITC apoptosis detection kit, Enzo Life Sciences) and stained using the ANNEXIN V-FITC apoptosis detection kit (Enzo Life Sciences) at room temperature for 5 min in the dark, according to the manufacturer’s instructions. Stained cells were analyzed using a Flow cytometer (BD Biosciences, Heidelberg, Germany) and the data obtained was analyzed using the FACS (Fluorescence Activated Cell Sorting)-Canto II software.
Cell cycle analysis
After treatment with MEKS for 4 h, HSC4 cells were trypsinized, harvested and washed with PBS twice. After homogenisation, cells were fixed with 75% ethanol and washed with PBS. They were then re-suspended in PBS containing RNase (40 μg/ml) for 30 min and treated with PI solution (10 μg/ml). Cells were then transferred to FACS tubes and DNA contents were analysed using a FACS Scan flow cytometer. Data were analysed using FACS-Canto II software.
Intracellular reactive oxygen species detection
The levels of intracellular (ROS) were measured using DCFH-DA. Briefly, HSC4 cells (2.5x104) were cultured overnight in a 96-well plate. Then, 50 μM of DCFH-DA was added and the cells were cultured for a further 60 min. Cells were then treated at the concentration of 0, 15, 30, 45, or 60 μg/ml of MEKS and fluorescence intensities were measured using a TECAN Infinite M200 fluorometric plate reader (excitation at 485 nm, emission at 530 nm, Munnedorf, Switzerland).
Western blot analysis
HSC4 cells were treated with MEKS at the concentration of 0, 25, 50 or 75 μg/ml for 4 h, then cultured in fresh media for further 20 h. After treatment, cells were collected and lysed with RIPA buffer (Cell Signaling Technology) for 30 min. Then, the supernatant was taken by 20 min centrifugation at 12,000 rpm, diluted in sodium dodecyl sulfate (SDS) buffer, and boiled for 5 min. After quantification, 50 μg of protein extract were subjected to electrophoresis using 10% SDS-polyacrylamide gel, then transferred to polyvinylidene fluoride membrane for 2 h. Blocking was performed using TNE buffer [50 mm Tris/HCl (pH 7.4), 100 mm NaCl, 0.1 mm EDTA] containing 5% skim milk and 0.1% tween-20. Antibodies against Bax, cleaved PARP, cleaved caspase 3, cleaved caspase 8 and cleaved caspase 9 were incubated overnight for 4 ℃. Then, it was applied with horseradish-conjugated secondary antibody and detected using SuperSignal West-Femto reagent (Pierce, Rockford, IL, USA).
Statistical analysis
The Student’s-t test in Window PASW (Predictive Analytics SoftWare) version 21.0 (SPSS Inc, Chicago, IL, USA) was used to analyse the significance of differences between the control and MEKS-treated groups. Results are presented as means ± standard errors and statistical significance was accepted for P<0.05, as indicated.
Ⅲ.RESULTS
MEKS treatment inhibited cell proliferation and modified the morphology of oral cancer cells.
MEKS inhibited cell proliferation in a dose-dependent manner (Figure 1a), with an IC50 value (50% growth inhibition) of 45.3 μg/ml. After treatment with MEKS, cells became smaller and rounder and lost their cellular processes (Figure 1b). MEKS-treated cells displayed the ruptured cell membrane without chromatin clumping or apoptotic body formation, distinguished from apoptotic characteristics. Taken together, these results demonstrated that MEKS inhibited cell proliferation and induced cell death.
MEKS induced early and late apoptosis in HSC4 cells
7-AAD and ANNEXIN V FITC double staining demonstrated that MEKS increased the number of apoptotic cells in a dose-dependent manner. In particular, treatment with of MEKS at 75 μg/ml caused the apoptosis of 78.5% of cells (Figure 2).
MEKS induced sub-G1 arrest and increased the number of apoptotic cells
Only 1.0% of control cells were in the sub-G1 phase. However, cells treated with 50 μg/ml MEKS showed a marked increase in the number of cells undergoing sub-G1 arrest. The proportions of sub-G1 arrested cells in the 50 and 75 μg/ml MEKS were 84.8% and 93.7%, respectively (Figure 3).
MEKS elevated intracellular ROS levels
Treatment with MEKS markedly increased intracellular ROS levels, as shown in Figure 4. ROS was found to accumulate in a time-dependent manner, with peak ROS production occurring following exposure to 25 and 100 μg/ml of MEKS. After 60 min of incubation with 25 μg/ml of MEKS, intracellular ROS levels were approximately 2.0 fold higher than in the untreated control group (Figure 4).
MEKS did not induce the intrinsic and extrinsic apoptotic pathway
Interestingly, treatment of HSC4 cells with MEKS did not induce intrinsic pathway-related proteins such as caspase 8 and extrinsic pathway-related proteins such as caspase 9, caspase 3 and cleaved PARP. In contrast, MEKS significantly increased the levels of Bax protein in a dose-dependent manner (Figure 5).
DPQ pretreatment alleviated cell death induced by MEKS treatment in HSC4 cells
To determine the specific type of cell death induced by MEKS treatment, we performed an MTT assay on cells pre-treated with DPQ (PARP1 inhibitor) for 1 h prior to MEKS treatment. It was found that the cell viability of the DPQ pre-treated HSC4 cells with MEKS treatment was significantly greater than that of MEKS treated-cells (Figure 6).
Ⅳ.DISCUSSION
Our results demonstrated that MEKS treatment caused the proliferation inhibition of HSC4 cells and the increase of late apoptotic cell numbers corresponding to increasing concentrations of MEKS. Moreover, the percentage of sub-G1 arrested cells and the production of ROS also increased. However, the cleavage of caspases including caspase 3, 8 and 9 were not detected in the MEKS-treated HSC4 cells. More importantly, the activation of Bax was detected and the application of DPQ effectively diminished the cell death in response to MEKS treatment. Taken together, these findings indicated that the cell death induced by MEKS occurs via apoptotic mechanisms.
Apoptosis is characterized by typical morphological features, including chromatin condensation, nuclear fragmentation, apoptotic body formation, cellular shrinkage, and plasma membrane blebbing5). In contrast, MEKS-treated HSC4 cells showed morphological changes in a somewhat different form from characteristic apoptotic morphology. The MEKS-treated cells displayed the ruptured cell membrane without chromatin clumping or apoptotic body formation. Furthermore, the cleavage of caspases involved in the apoptotic pathway was not detected. DPQ pretreatment with MEKS abolished MEKS-induced cell death. Programmed necrosis is becoming increasingly realized as cell death that can take place through regulated mechanisms or pathways, termed necroptosis23). This type of cell death occurrs via the activation of tumor necrosis factor (TNF) alpha, Fas L, and TRAIL, which are the same proteins that can trigger an apoptotic pathway23). However, in the case of inactivation of caspases, the alternative cell death pathway, necroptosis, can be stimulated.
RIP kinases are crucial regulators of cell survival and cell death, and required as key regulators of necroptosis. Our results demonstrated DPQ pretreatment prior to MEKS decreased cell death effectively. In addition, the cleavage of caspases which are essential in the apoptotic pathway did not occurr. Given the different cell morphology, the cell death induced by MEKS treatment in HSC4 cells seems to be necroptosis.
ROS are produced as a natural byproduct of the normal metabolism of cells and play an important role in cell signaling and homeostasis24). It involves various cellular processes, physiological conditions as well as pathological conditions. Under pathological conditions like ionizing radiation, heat exposure, and UV, uncontrolled excessive ROS production begins and provokes various cellular responses. Mitochondrial ROS can intercede cell death mechanisms and it shows characteristic ultrastructural changes3,25). The necroptosis pathway is closely connected with ROS production and mitochondrial as well as non-mitochondrial3). Irrinki et al. reported that the over-expression of antioxidants diminished ROS production and protected cells from necroptosis26). Therefore, similar to apoptosis, ROS plays an essential role in the initiation of the necroptic pathway.
Bax is a crucial regulator during apoptotic cell death, especially mitochondria-dependent apoptosis. It mediates not only the apoptosis by an intrinsic pathway, but also a necroptosis pathway through mitochondrial membrane permeabilization27). The activated Bax protein can induce the translocation in mitochondria, and trigger a cytochrome C release28,29). In the necroptotic pathway, MEK/ERK and JNK can be triggered by DNA damage and result in necrotic cell death30,31). However, the regulatory mechanism by the activation of Bax protein can be related to specific cell types and depends on cytotoxic agents.
Between PARP1 activation and ROS production, there are bidirectional interactions32). ROS production can trigger PARP1 activation, and PARP1 activation may induce ROS production form mitochondria33). Chen et al. reported that ROS production and PARP activation eventually led to necroptosis in murine L929 fibrosarcoma cells34). Dysfunction of mitochondria plays an essential role in PARP-induced necroptosis, proven by the PARP1 inhibitor (DPQ) pretreatment25).
Recently, several approaches demonstrated that necroptosis can be a novel strategy for cancer chemotherapy35-38). The research indicated that induction of necroptosis is a valuable cancer strategy in several cancer cells. More importantly, necroptosis seems to be a promising strategy under conditions where apoptosis fail or not be effective2,35). These days, it is promising to discover that new chemotherapeutic agents can implicate multiple cell death pathways. Understanding the mechanism of necroptosis and its application in new therapeutic agents for cancer can be an alternative strategy.
In summary, the results of our study revealed the induction of necroptosis in HSC4 cells by MEKS treatment. Additionally, MEKS can be a promising adjuvant chemotherapeutic agent that induces multiple cell death pathways.
