Orostachys japonicus induce p53‐dependent cell cycle arrest through the MAPK signaling pathway in OVCAR‐3 human ovarian cancer cells

Abstract Orostachys japonicus (O. japonicus) is utilized as a traditional medicine for patients with various diseases. This study investigated the effect of the ethyl acetate fraction from O. japonicus extract (OJE) on the growth inhibition of OVCAR‐3 human ovarian cancer cells demonstrated to inhibit cell growth and arrest the cell cycle in OVCAR‐3 cells by blocking the sub‐G1 phase and decreasing cyclin E1/CDK2 expression. Cell cycle arrest was connected to the increased expression of the cell cycle regulating factors p53 and p21. Apoptosis was initiated through the intrinsic pathway by up‐regulating the expression of the Bcl‐2/Bax ratio and down‐regulating the expression of pro‐caspase‐3. Furthermore, OJE treatment elicited p‐p38 activation and p‐ERK1/2 inhibition. In conclusion, our results demonstrated that OJE reduced the growth of OVCAR‐3 human ovarian cancer cells mediated by arrest of the cell cycle and regulation of MAPK signaling pathways.

Secondary antibodies, an Annexing V-FITC assay kit and cell cycle assay kit were purchased from BD Pharmingen™ (BD Biosciences, USA).

| Preparation of OJE fraction from O. japonicus
Dried O. japonicus powder was supplied by Geobugiwasong Ltd. (Miryang, Korea). The ethyl acetate (EtOAc) fraction from O. japonicus was fractioned using a solvent, as described by our team (Lee et al., 2014;Ryu et al., 2012). The EtOAc fraction was concentrated by evaporation at 40°C to achieve dryness, and stored in dimethyl sulfoxide (DMSO) at −20°C.

| GC-MS analysis
Component analysis of the EtOAc fraction (OJE) has previously been described by our team (Lee et al., 2014;Ryu et al., 2012).

| Cell viability assay
Cell viability was determined with a CellTiter 96 AQueous One Solution Cell Proliferation Assay Kit (Promega Corporation, Madison, WI, USA) according to the manual. OVCAR-3 cells were incubated with serial concentrations (0, 12.5, 25, 50 μg/ml) of OJE for 24 hr. After incubation, 10 μl of MTS solution was added to the well and incubated for 3 hr. The absorbance in the wells was measured at 490 nm using a FilterMax F5 Multi-Mode microplate reader (Molecular Devices, USA).

| Quantification of apoptosis by flow cytometry
OVCAR-3 cells were treated with OJE for 24 hr and harvested with 0.25% trypsin-EDTA treatment. The apoptotic cells were detected using 10 μl of annexin V-FITC and 5 μl of propidium iodine (PI) for 15 min in the dark (BD Biosciences, USA) and then analyzed with a FACSCalibur flow cytometer (BD Biosciences, USA). For each condition, populations of 1 × 10 4 cells were determined in each cytometry experiment.

| Cell cycle analysis
Cells (5 × 10 5 /ml) were plated in six-well plates followed by treatment with OJE for 48 hr. The cell cycle phase was assayed by DNA fragment staining with PI solution using a cell cycle phase detection kit (BD Bioscience, USA). Cells were determined by FACSCalibur flow cytometry (BD Biosciences, USA).

| Detection of apoptotic body by DAPI staining
The apoptotic bodies were stained using the 1 μg/ml DAPI solution (Vector Laboratories, USA) according to the manufacturer's instructions. Cells were treated with OJE fraction for 24 hr.
After incubation, the cells were washed with cold PBS and then fixed in cold 4% paraformaldehyde for 30 min. Apoptotic bodies were dyed blue and fixed with mounting medium. After staining, cells were analyzed using fluorescence microscopy on AMG (Washington, USA).

| RNA extraction and Reverse Transcription PCR
Cells were treated with different concentrations (0, 12.5, 25, 50 μg/ml) of OJE for 24 hr. Total RNA was isolated using the Trizol reagent (Invitrogen, USA). The concentration and purity of the RNA were measured by a FilterMax F5 Multi-Mode microplate reader (Molecular Devices, USA). cDNA was synthesized using 1 μg of total RNA per 20 μl of reaction mixture using AccuPower RT PreMix reagent for the reverse transcription (Bioneer, Korea).
Target gene duplication was performed using specific oligonucleotide primers of right and left in the PCR system. The primer sequences and conditions used in the PCRs are listed in Table 1. The PCR products were electrophoresed on agarose gels and stained using ethidium bromide (EtBr). The bands were determined and visualized using the Davinch-Chemi™ imaging system (Davinch-K, Korea).

| SDS-PAGE and Western Blot analysis
OVCAR-3 cells were treated with OJE for appropriate times. The cells rinsed twice with PBS and lysed using cell lysis buffer (Cell Signaling Technology, USA) containing a protease inhibitor cocktail in six-well plates (Roche Diagnostics Ltd., Germany). The total concentration of protein was measured using a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, IL). To quantify the expression of the target protein, the sample was subjected to 8%-10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The membrane was blocked with 5% BSA, incubated with primary antibody (all antibodies were diluted at 1:1,000 in 3% BSA) for 24 hr at 4°C and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (the antibody was diluted at 1:10,000 in 3% BSA) for 2.5 hr. The protein bands on the membrane were visualized using enhanced chemiluminescense (ECL) solution (Santa Cruz Biotechnology, USA), and signals were detected using the Davinch-Chemi ™ imaging system (Davinch-K, Korea).

| Statistical analysis
The results are expressed as mean ± standard deviation (SD) and analyzed by SPSS version 22.0 (SPSS Inc., USA). p < 0.05 was considered significantly.

| RE SULTS AND D ISCUSS I ON
In this study, the intracellular signaling pathways of OJE-induced apoptosis in OVCAR-3 cells were investigated. Orostachys japonicus was extracted sequentially by organic solvents, including ethyl alcohol (EtOH), n-hexane (Hex), dichloromethane (DCM), ethyl acetate (EtOAc), n-butanol (BuOH), and water (H 2 O). Among these, the EtOAc fraction was the most active against OVCAR-3 cancer cells. This fraction was analyzed using a GC-MS system following the measurement methods detailed in our study. As a result, the following three peaks were identified: "gallic acid (4.24%), kaempferol (6.81%), and quercetin (5.08%)" (Lee et al., 2014;Ryu et al., 2012). Various studies have revealed that kaempferol and quercetin lead to apoptosis using the natural products in various human cancer cells (Lee, Szcsepanski, & Lee, 2008;Yoshida et al., 2008). However, many O. japonicus ingredients have not yet been identified and warrant further study to determine their various physiological activities.

| OJE inhibits cell proliferation in OVCAR-3 cells
An anticancer effect commonly involves mechanisms that induce apoptosis and abnormal cell growth inhibition. The suppression of abnormally proliferating cancer cells is an important screening result factor that proves the anticancer activity of drugs (Li et al., 2015). To confirm the effects of

| Effect of OJE on the intrinsic apoptotic and upstream signaling pathway
Apoptosis has two pathways of extrinsic (or death receptordependent) and intrinsic (or mitochondrial-dependent) factors. The intrinsic pathway is regulated by pro-apoptotic and anti-apoptotic proteins, such as Bax, Bak, Bid, Bcl-2, and Bcl-xL. In particular, the interaction between Bax and Bcl-2 leads to a loss of mitochondrial membrane potential. As a result, mitochondrial cytochrome c is released into the cytoplasm from the mitochondria and activates the caspase pathways (Dong et al., 2014;Kassi et al., 2009). In our study, OJE-treated cells did not exhibit dramatically altered Bax mRNA levels, but the Bcl-2 levels were certainly decreased at the highest treatment concentration (50 μg/ml). As a result, the expression of procaspase-3 decreased, leading to the activation of apoptosis in OVCAR-3 cells.
Mitogen-activated protein kinases (MAPKs) are involved in the upstream signaling pathway of various cell phenomena, such as proliferation, development, differentiation, transformation, and apoptosis. The MAPKs families consist of three subfamilies, namely ERK, JNK and p38 (Romos, 2008;Zhang & Liu, 2002). Numerous studies have focused on ERK1/2 inhibition leading to cancer cell death (Lunghi et al., 2003;Wang et al., 2010;Wu, Wong, Khosravi, Minden, & Penn, 2004). Furthermore, p38 MAPK plays a role in cell cycle arrest by activating cell cycle checkpoints (Feng et al., 2015;Thornton & Rincon, 2009). To determine the MAPK signaling pathways, we examined the activity of subfamilies such as ERK1/2 and JNK as well as p38 phosphorylation using western blot analysis. As shown in Figure 4, the expression of phosphorylated ERK1/2 (p-ERK1/2) was significantly reduced and phosphorylated p38 (p-p38) was gradually increased. The effective combination of p-ERK1/2 and p-p38 is expected to act in concert as a positive apoptosis mediator in OJE-treated OVCAR-3 cancer cells.

| CON CLUS ION
In conclusion, in our study OJE was found to produce anticancer effects via the induction of apoptosis and cell cycle arrest in OVCAR-3 cells. Sub-G1 cell cycle arrest induced by OJE occurred through cyclin E1/CDK2, p21, and p53-mediated pathways. Furthermore, the down-regulation of Bcl-2 and pro-caspase-3 indicated that the apoptosis signaling pathway was mediated through mitochondria.
Upstream MAPKs signaling pathways play important roles in the OJE-induced anticancer activity observed in OVCAR-3 cancer cells.
These results suggest that OJE is expected to be used as a powerful anti-cancer drug derived from nature in OVCAR-3 human ovarian cancer cells.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.