Anti-Proliferative Effects of Siegesbeckia orientalis Ethanol Extract on Human Endometrial RL-95 Cancer Cells

Endometrial cancer is a common malignancy of the female genital tract. This study demonstrates that Siegesbeckia orientalis ethanol extract (SOE) significantly inhibited the proliferation of RL95-2 human endometrial cancer cells. Treating RL95-2 cells with SOE caused cell arrest in the G2/M phase and induced apoptosis of RL95-2 cells by up-regulating Bad, Bak and Bax protein expression and down-regulation of Bcl-2 and Bcl-xL protein expression. Treatment with SOE increased protein expression of caspase-3, -8 and -9 dose-dependently, indicating that apoptosis was through the intrinsic and extrinsic apoptotic pathways. Moreover, SOE was also effective against A549 (lung cancer), Hep G2 (hepatoma), FaDu (pharynx squamous cancer), MDA-MB-231 (breast cancer), and especially on LNCaP (prostate cancer) cell lines. In total, 10 constituents of SOE were identified by Gas chromatography-mass analysis. Caryophyllene oxide and caryophyllene are largely responsible for most cytotoxic activity of SOE against RL95-2 cells. Overall, this study suggests that SOE is a promising anticancer agent for treating endometrial cancer.

concentrations under different incubation durations. Experimental results indicate that SOE inhibited cell viability in dose-and time-dependent manners ( Figure 1A,B).

Apoptotic Effects of SOE on RL95-2 Cells
Apoptosis, a programmed cell death, plays a critical role as a protective mechanism against carcinogenesis by eliminating unnecessary or unwanted cells to maintain a healthy balance between cell survival and cell death [19]. The relationship between apoptosis and cancer has been a recent focus. Apoptosis provides many valuable clues about a therapy's effectiveness, and hence the anticancer effects of many chemotherapeutic agents are via apoptosis [20]. Therefore, induction of apoptosis has become a principal focus when developing anticancer therapies.

Cell Morphology
Apoptosis can be characterized by particular morphological changes, such as plasma membrane bleb, cell shrinkage, chromatin condensation and DNA fragmentation [21]. Figure 1C shows the morphological changes of RL95-2 cells after SOE treatment for 24-72 h. Phase-contrast micrographs reveal that SOE induced typical morphological characteristics of apoptosis, including cell shrinkage, apoptotic vacuoles, membrane blebbing and formation of floating cells, in a dose-and time-dependent manner. These changes are in agreement with findings in literature on apoptosis [21][22][23].

Cell Cycle Regulation
To determine whether SOE induced the arrest of cell cycle progression in RL95-2 cells, flow cytometry was applied to quantitate the cell cycle distribution under treatment with different SOE concentrations (0-150 μg/mL) for various durations (24-72 h). The number of cells in the G2/M phase increased significantly, and that in the G0/G1 phase decreased in SOE-treated cells as the SOE dose increased during 48-72 h treatment ( Figure 2). Moreover, this effect also increased over treatment time. This experimental finding implies that SOE could arrest RL95-2 cells at G2/M phase.
The Annexin V-FITC apoptosis detection kit was then used to examine the effect of SOE on RL95-2 cell death by flow cytometry. Figure 3 shows that the cell dots were dispersed and shifted to the lower right side, indicating that the cells moved to the early apoptotic stage. When the SOE dose or treatment time increased, the number of cell dots in the upper right side increased, implying that cells progressed gradually to the late apoptotic stage. Conversely, the number of cell dots on the upper left side was not significantly changed, implying that only few cells died via necrosis. These experimental results demonstrate that SOE induced apoptosis of RL95-2 cells.

Pro-Apoptotic and Anti-Apoptotic Proteins Expression
Apoptosis can occur through two fundamental pathways: (1) the mitochondrial or intrinsic pathway; and (2) the death receptor or extrinsic pathway [24]. Caspase-9 and -8 are the protease indexes for the intrinsic and the extrinsic pathway, respectively. Caspase-3, a well-known downstream adaptor caspase, can be activated by caspase-9 or -8 via the intrinsic or extrinsic signaling pathways [20]. To elucidate the molecular effector pathway of SOE-mediated apoptosis, this study determined whether caspases are involved as downstream effectors. As shown in Figure 4, SOE increased cleavage of procaspase-3, -8 and -9, accompanied by an increase in caspase-3, -8 and -9 expression in a dose-dependent manner. This implies that apoptosis was through the intrinsic and extrinsic pathways.     Several cytoplasmic proteins are involved in regulation of apoptosis; particularly members of the Bcl-2 family; one subgroup, including Bcl-2 and Bcl-xL, inhibits apoptosis, while the other, including Bad, Bak and Bax, promotes cell death [20,25]. Since SOE induces the apoptosis of RL95-2 cells, the effect of this extract on cellular proteins that are involved in apoptosis was examined. As the SOE concentration increased, the expressions of Bad, Bak and Bax proteins increased, while those of the Bcl-2 and Bcl-xL proteins decreased ( Figure 4). These experimental findings suggest that SOE induced apoptosis of RL95-2 cells.

Cytotoxicity of SOE on Various Cancer Cell Lines
To investigate the potential effect of SOE against other cancers, various cancer cell lines were used to assess the cytotoxicity of SOE and quantified using the MTT assay. Notably, as shown in Table 1, SOE was effective against all these cell lines, and particularly on LNCaP human prostate cancer cells (IC50 = 87.2 ± 1.3 μg/mL under 24 h treatment).
Both CPO and CP are sesquiterpenes isolated mainly from the essential oils of such medicinal plants as Ocimum basilicum, Tagetes minuta [26], Toona sinensis [27], Hyptis spicigera and Lippia multiflora [28]. Caryophyllene had anti-proliferative ability on many cancer cell lines, including oral, liver, lung, colon, melanoma, leukemia and erythroleukemia [29][30][31]. Notably, CPO inhibited growth and induced apoptosis through suppression of the PI3K/AKT/mTOR/S6K1 pathways and ROS-mediated MAPKs activation in human prostate and breast cancer cells [32,33]. According to Kim et al. [34], CPO retarded proliferation, induced apoptosis and abrogated the invasion by suppressing constitutive and inducible STAT3 activation in multiple myeloma, breast and prostate cancer cell lines. Therefore, these two ingredients may be responsible for the cytotoxicity of SOE against human endometrial carcinoma cell line RL95-2.  Relative abundance (−) have higher cytotoxicity than CP, on human T lymphocyte Jurkat cells and human neuroblastoma IMR-32 cells, from a structural biology in silico model based on the degree of stability of the complexes formed between arachidonate 15-lipoxygenase and two caryophyllenes [35]. Additionally, Oh et al. reported that CPO had a higher acaricidal activity against house dust mites than CP [36]. These studies revealed that a slight structural difference of CP may significantly affect their bioactivities.

Preparation of S. orientalis Ethanol Extract
The aerial part of S. orientalis L. was freeze-dried and ground into powder. The dried powder (9.3 kg) was extracted with 47 L of 95% ethanol by stirring at room temperature for 1 day; this was repeated 3 times. The extracted solutions were collected and filtered through filter paper (Whatman No. 1; Whatman Paper Ltd, Maidstone, Kent, UK). The SOE was obtained by removing solvent with a rotary evaporator and dried it in a freeze-drier. Total dry weight of this extract was 489 g (extraction yield = 5.3%). In cell culture test, the SOE was dissolved in dimethyl sulfoxide (DMSO) and the final DMSO concentration was less than 0.1% in medium.

Determination of Cytotoxicity for Cancer Cells
Cancer cells were cultured in 96-well plates at 1 × 10 4 cells/well, treated with the indicated concentration of SOE, and cultivated in 100% relative humidity of 5% CO2 and 37 °C. After specified cultivation duration, the medium solution was removed. An aliquot of 100 µL culture medium containing 0.5 mg/mL of MTT assay kit was loaded onto the plate. The cells were cultured for 2 h, and then the medium solution was removed. An aliquot of 100 µL DMSO was added and the plate was shaken until its crystals dissolved. The cytotoxicity against cancer cells was determined by measuring the absorbance of the converted dye at a wavelength of 570 nm with an ELISA reader (Model 550, Bio-Rad Laboratories, Hercules, CA, USA). Cytotoxicity of each sample is expressed as IC50 value, which is the concentration of test sample that cause 50% inhibition or cell death, and was obtained by plotting the percentage inhibition versus SOE concentration.

Flow Cytometry Analysis of Cell Cycle
The effect of SOE on cell cycle distribution was investigated by flow cytometry after staining the cells with propidium iodide (PI). The RL95-2 cells (2 × 10 5 cells/well) in 24-well plates were treated with various concentrations of SOE and cultured for 24, 48 or 72 h. The treated cells were harvested and washed with phosphate-buffer saline (PBS). Next, 100 μL Trypsin-EDTA solution were added to detach the cells. After centrifugation at 100 g for 5 min, the cell pellet was suspended with 70% ethanol and kept at −20 °C for 12 h. Then, the cells were washed with cold PBS and suspended in PBS containing 20 μg/mL PI, 0.2 mg/mL RNase A and 0.1% Triton X-100 at 4 °C for 12 h. The stained cells were then analyzed by flow cytometer (FACSCalibur System, BD Biosciences) and the data were calculated with WinMDI software (Version 2.9, TSRI, La Jolla, CA, USA).

Apoptotic Ratio Analysis
The apoptotic effect of SOE on RL95-2 cells was determined by Annexin V-FITC staining method and measured using flow cytometer. The RL95-2 cells (2 × 10 5 cells/well) in 24-well plates were treated with various SOE concentrations and cultured for 48 h. The treated cells were harvested and washed with PBS. Next, 100 μL Trypsin-EDTA solution was added to detach the cells. After washing with cold PBS, the cells were centrifuged at 200 g for 10 min. The cell pellet was suspended in 100 μL of Annexin V-FITC staining-solution and incubated for 20-30 min at 25 °C. The cells were then analyzed by flow cytometry.

Western Blot Analysis
To analyze the proteins of procaspase-3, procaspase-8, procaspase-9, Bcl-family, 1 × 10 5 cells were seeded into 6-cm culture dishes with or without SOE, and were incubated for 24 h. The medium was removed and cells were washed several times with PBS (0.01 M, pH 7.2). Whole-cell lysates were prepared using the procedures described earlier. The harvested protein concentration was measured using a protein assay kit (Bio-Rad). The same amounts of proteins from each extract were applied to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred onto a nitrocellulose membrane (Immunobilin P; Millipore, Billerica, MA, USA), and were then blocked by 10% skim milk in water for 1 h. After washing three times with PBS containing 0.1% Tween-20, the specific primary antibodies with a suitable dilution were added. Following overnight incubation at 4 °C, the primary antibodies were washed away and secondary antibodies were added for 1 h incubation at room temperature. The protein levels were determined by using Enhanced Chemiluminescence (ECL) Plus Western blotting detection reagents (Amersham Bioscience, Uppsala, Sweden) to develop the signal of the membrane. Densitometric analyses were conducted using the Quantity One ® software (Bio-Rad).

Gas Chromatography-Mass Spectrometry
The GC-MS analysis was performed using Varian 450-GC and 240-MS system (Varian, Salt Lake City, UT, USA) with the electron impact mode (70 eV) injector, and a Varian data system. The GC column was VF-5ms capillary column (30 m × 0.25 mm, film thickness 0.25 μm, FactorFour TM , Varian, Salt Lake City, UT, USA). Injector and detector temperatures were set at 250 °C and 290 °C, respectively. Oven temperature was kept at 50 °C for 5 min, then raised to 120 °C by a rate of 5 °C/min, kept at 120 °C for 8 min, then raised to 300 °C by a rate of 10 °C/min. The carrier gas was helium at a flow rate of 1 mL/min. Diluted samples of 1.0 μL was injected under the splitless mode. The percentages of the ingredients were calculated by the area normalization method. The components were identified by comparison of their mass spectra with the NIST MS 2.0 database (Gaithersburg, MD, USA).

Statistical Analysis
All experiments were carried out for three to five independent replicates. The experimental data were analyzed by using Microsoft Excel software (Microsoft Software Inc., Redmond, WA, USA). The data are expressed in terms of mean and standard deviation, and the statistical differences were analyzed by Student's t-test (* p < 0.05, ** p < 0.01, *** p < 0.001).

Conclusions
This study demonstrates that SOE had significant cytotoxicity directly towards RL95-2 endometrial cancer cells. Under SOE treatment, the cell morphology change, cell cycle regulation, apoptosis by Annexin V-FITC detection, and expressions of pro-apoptotic and anti-apoptotic proteins expression of RL95-2 cells were investigated. Notably, SOE induced apoptosis of RL95-2 cells via both intrinsic and extrinsic signaling pathways. Further, CPO and CP were responsible for most of the cytotoxicity of SOE. Taken together, these experimental findings suggest that SOE is a potential source for both the prevention and treatment of endometrial cancer. Further studies are required to identify the bioactive components and their mechanisms of action.