1. Introduction

Due to rapid industrial and economic development, irregular lifestyles, lack of exercise, and pollution, the rate of chronic diseases includes cancer, cardiovascular diseases, and mental illnesses is increasing each year. Among these chronic diseases, cancer is the primary cause of death globally. According to the updated data from cancer statistics in Korea in 2015, colon cancer is the third(12.13%) leading cause of death in Korea[1]. Although surgery,chemotherapy, and/or radiation therapy are used as conventional cancer treatments, these methods may lead to adverse side effects such as DNA damage, neural toxicity, and hair loss, which possibly hinder the successful treatment of the disease. Many recent studies have been conducted on natural substances that are safer and more effective than conventional methods. Apoptosis is an essential step in cell proliferation and the maintenance of homeostasis in multicellular organisms, blebbing, cell shrinkage, and DNA fragmentation[2-4]. Two well-known mechanisms of apoptosis are the mitochondria-mediated intrinsic apoptotic pathway and the death receptor mediated extrinsic apoptotic pathway. Inducing apoptosis in cancer cells has been thought to be crucial to the development of organisms and maintenance of tissues, as it leads to selective cell death. The initiation of apoptotic signaling pathway suppresses anti-apoptotic proteins for example B cell lymphoma-2 (bcl-2)or bcl-XL, and then activates bcl-2-associated x protein (bax) or bcl-2 homologous antagonist killer (bak). Bax is a pro-apoptosis protein that helps to promote apoptosis as it begins, whereas bcl-2 is an anti-apoptotic protein. Decreasing the expression of bcl-2 has been shown to dramatically reduce the number of leukemic cells,making it a potentially very useful way for treating cancer[5-7]. In the intrinsic pathway, cytochrome c released from mitochondria binds pro-caspase-9 to activate it, and then, the activated cleaved caspase-9 then activates the effector pro-caspase-3 into cleaved caspase-3 which degrades cells[8-12]. Mitogen activated protein kinase (MAPK)is also known to be participated in induction of apoptosis according to several studies[13,14].

Orostachys japonicus (O. japonicus), a biennial herb plant which belongs to the family Crassulaceae and named as Wa-song in Korea, is used conventionally as a folk medicine for its activities of anti-inflammation, anti-febrility, hemostasis, antidote, and especially anti-cancer. Friedelin, epifriedelanol, glutinone, glutinol,β -amyrin, triterpenoid derivatives, campesterol, sterol glucosides,fatty acid esters, kaempferol, quercetin, flavonoid glycosides,1-hexatriacontanol, and oxalic acid have been reported to be contained in this herb[15-23]. Furthermore, it has been shown to be effective in treating stomach cancer, liver cancer, and colon cancer and to have anti-microbial and anti-diabetic substances. In our studies, dried milled O. japonicus was collected with ethanol and categorized with organic solvents[20]. After identifying the ethylacetate fraction through gas chromatography-mass spectrometry, 12 peaks were taken. Among them, three peaks were identified as kaempferol (6.81%), quercetin (5.08%), and gallic acid(4.24%)[20,21]. These soluble fractions were examined for activities of anti-cancer, anti-inflammatory, and anti-oxidizing activities.Among these extracts, ethylacetate fraction exhibited the best activity for anti-cancer. Although previous reports have disclosed the effectivity of ethylacetate fraction in treating stomach cancer and liver cancer[19-24], its roles in induction of necessary for apoptosis,the cell cycle block, and intracellular signaling pathways in HT-29 colon cancer cells have remained uninvestigated. In this study, we provide some solid evidences to demonstrate the mechanism of the anti-colon cancer activity of the ethylacetate fraction derived from O.japonicus.

2. Materials and methods

2.1. Cell Line and reagents

The ethylacetate fraction was extracted from O. japonicus (OJEF for short) in our laboratory using methods described previously[20- 25].HT-29 cells were gained from the Korean cell line bank (KCLB;Seoul, Korea). DMEM, 10% FBS, antibiotic solution, monoclonal antibodies such as bcl-2, bax, caspase-3, -8, -9, cleaved caspase-3, -8, -9, phospho-MAPK, MAPK, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and secondary antibody against goat anti-rabbit IgG-HRP were prepared from the same suppliers as described previously[20-25].

1.3.1 设备 飞利浦大孔径的螺旋CT，网状热可塑体膜，瓦里安600C/D及23EX的直线加速器，瓦里安Eclipse的治疗计划系统。

2.2. Cell culture

The HT-29 cells were sub-cultured once 5 days. The medium was replaced every 3 days. When cultures were 85%-95% confluent,they were used for experiments. The OJEF solution mixed with 0.1%dimethyl sulfoxide (Sigma-Aldrich, MO, USA) was used to treat the cells.

2.3. Cell viability assay

The CellTiter 96 Non-radioactive Cell Proliferation Assay Kit(Promega, WI, USA) was used to assess cell viability. The cells were seeded in 96-round well plate and treated with OJEF. After complete removing of the medium and washing with PBS, and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) reagent was added. The plate was placed at 37 ℃ for 1 h, and then, the parameter was analyzed at 490 nm using an ELISA reader (PowerWaveXS, BioTek, VT, USA).

首先对论证区域1980—2011年进行补给量和排泄量的计算，经计算本论证区域多年平均地下水资源量1 378.3万m3。2015规划水平年开采量1 369.3万m3，开采量占地下水资源量的99.3%；2020规划水平年开采量1 363.8万m3，开采量占地下水资源量的98.9%。

2.4. Flow cytometric analysis of apoptosis

An Annexin V-FITC Apoptosis Detection kit (BD Biosciences, NJ,USA) was used to assess the apoptotic rate of HT-29 cells. The cells were seeded in 24-round well plate and treated with OJEF. After 12 h, the medium was completely removed and washed. Binding buffer was added, subsequently applying with annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) reagent. The reaction was allowed to take place in the dark for 15 min, and then the samples were analyzed using flow cytometry (Becton Dickinson, NJ,USA).

2.5. Flow cytometric analysis of cell cycle

The HT-29 cells were dispensed into 6-round well plate and then treated with OJEF. Cold PBS was completely removed, and the proteins were collected using lysis buffer. Protein concentrations were calculated using a BCA protein Assay kit (Thermo scientific,IL, USA). Testing materials were prepared by mixing the quantified proteins and loading buffer in a 1：1 ratio and heating them at 95 ℃for 5 min. The samples prepared at different concentrations were electrophoresed on a 10%-15% SDS-PAGE, and then transferred to a PVDF membrane using a semi-dry method (Bio-Rad, CA,USA). The membrane was blocked using 5% non-fat milk at 4 ℃for at least 4 h, and then incubated with a primary antibody at 4 ℃ overnight. The membrane was then washed with PBST thrice and treated with a secondary antibody for approximately 2 h. The fluorescent protein bands were prepared with a luminol reagent, and developed in a darkroom using a film.

2.6. Nuclear staining assay

The HT-29 cells incubated for 12 h were suspended in Triton-X-containing PBS solution. They were centrifuged; the cell pellets were suspended in 4',6-diamidino-2-phenylindole (DAPI; Vector Laboratories, CA, USA) staining solution. Subsequently, 20 μL of the suspension was dropped on glass slide and covered with a cover glass, such that the bubbles were avoided. This was incubated at cold temperature overnight. Morphological changes in the nucleus were checked by fluorescence microscope (LSM510 Meta, Carl Zeiss,Jena, Germany).

2.7. Western blotting

A Cell Cycle Phase Determination kit (Cayman Chemical, MI,USA) was used to assess the cell cycle division. The HT-29 cells were dispensed into 24-round well plate and then treated with OJEF.After 12 h, the medium was completely removed and washed. Then,the assay buffer was applied. The cells were fixed using 100%ethanol for at least 2 h. The fixation solution was then completely removed, and the cells were suspended in a sufficient amount of RNase-containing PI staining solution. The reaction was allowed to occur in the dark place. The samples were analyzed by flow cytometry.

2.8. Statistical analysis

All experiments were carried out at least 3 times. The statistical differences were calculated in the Student's t test using the SigmaStat software (SigmaStat for Windows Version 3.0.). P＜0.05 was regarded statistically meaningful.

3. Results

3.1. Inhibition of cell growth

The proliferation of HT-29 cells treated with different concentrations of OJEF was assayed with MTS method. The HT-29 cells were treated with OJEF at concentrations of 25, 50, 75, and 100 μg/mL and incubated for 12 and 24 h. After 12-hour incubation,the respective cell viabilities were (66.84±6.30)%, (58.79±15.70)%,(34.86±5.70)%, and (30.02±6.20)%. After 24-hour postincubation,the respective cell viabilities were (62.94±21.13)%, (46.77±32.20)%,(31.08±29.71)%, and (17.19±19.21)%. The proliferation of OJEF-treated HT-29 cells decreased in a both concentration- and timedependent manner. These results show that OJEF suppress the proliferation of HT-29 cells.

首先，改变了资源稀缺性特点。在媒介融合背景下，网络信息技术、通讯技术以及数字技术三者在相互交融和发展中极大地缩小了媒介之间的壁垒，媒介融合促进市场一体化的形成，这样传统媒介信息传递过程中信息和渠道稀缺的现象不复存在。如今的媒介竞争将受众的喜好以及紧缺的市场资源作为重要的竞争区域。

3.2. Induction of apoptosis

此前，公司涉嫌信息披露违法违规，中国证监会决定对公司立案调查。披露违法强制退市情形，公司股票交易被实行退市风险警示。实行退市风险警示三十个交易日期限届满后，公司股票将被停牌，直至深圳证券交易所在十五个交易日内作出是否暂停公司股票上市的决定。二级市场上，该股近期走势维持强势震荡，但该消息对于股价后市增加了不确定性，后市注意风险。

Figure 1. Morphological changes in nucleus of HT-29 cells after being treated with ethylacetate fraction from O. japonicus. HT-29 cells were treated with 0.1% DMSO (control), 25, 50, 75, and 100 μg/mL OJEF (A, B, C, D, and E). Apoptotic bodies are showed by yellow arrows.

3.3. Induction of cell cycle arrest

To determine if there is a correlation between the anti-proliferation effect of OJEF on HT-29 cell growth and the apoptotic effect, we examined the cell cycle using flow cytometry. In the sub-G1 phase,which is an evidence of apoptosis (Table 1), treatment with 100 μg/mL of OJEF resulted in a (35.73±5.57)% arrest, in contrast to the control,which showed a (1.03±0.81)% arrest, thereby confirming an arrest in cell cycle progression. Similarly, with regards to the S phase,treatment with 100 μg/mL of OJEF resulted in a (11.68±2.36)%arrest, in contrast to the control, which showed a (5.20±1.15)%arrest, thereby confirming the increased impediment of cell cycle advance. The S phase cell proportion of the OJEF-treated cells increased in a concentration-dependent way. The HT-29 cells treated with OJEF caused manifest block of cell cycle in the sub-G1 and S phase.

Table 1 Effect of ethylacetate fraction from O. japonicus on cell cycle arrest in HT-29 cells.

The results are shown as the mean±SD. Cells treated only with 0.1%DMSO was used as control. *P＜0.05 was regarded statistically meaningful(vs. control).

3.4. Apoptosis-related downstream signaling pathways

Among the pro-apoptosis and anti-apoptosis factors, the correlation between the representative apoptosis-inhibiting factor bcl-2 and apoptosis-promoting factor bax was confirmed by their expression levels, which were measured by Western blotting. Table 2 shows bcl-2 decreased while bax increased in an OJEF concentrationdependent way. These results indicate that OJEF promoted apoptosis in HT-29 cells through the mitochondria-mediated pathway. Caspase proteins are integral proteases that induce cell apoptosis in relation to bcl-2 and bax proteins in downstream signaling pathways. Caspases are divided into three types according to structural differences：caspase-3, caspase-8, and caspase-9, and their roles also differ.To analyze the expression level of caspases during the cascade of activation Western blotting was executed. Cells were treated with concentrations of OJEF [0 (0.1% DMSO, control), 25, 50, 75, and 100 μg/mL] for 12 h. The expression of inactive forms such as procaspase-3, pro-caspase-8, and pro-caspase-9 decreased in the OJEF treatment group except 50 μg/mL OJEF group (Table 2). In contrast,the expression of the active forms of cleaved caspase-3, cleaved caspase-8 and cleaved caspase-9 increased in the OJEF treatment group except cleared caspase-8 in 100 μg/mL OJEF group.

3.5. Apoptosis-related upstream signaling pathway

To check if there is a correlation between the downstream mitochondria-mediated apoptosis pathways and upstream signaling pathways, we confirmed the MAPKs that are involved in directing the upstream signaling transduction system related to apoptosis using Western blotting. As shown in Table 3, the phospho-extracellular signal regulated kinase 1/2 (ERK1/2), Jun-N-terminal kinase (JNK),and p38 increased compared with the total form. In contrast, the total form of ERK1/2, JNK, and p38 did not change.

4. Discussion

In this study, experiments were performed to investigate how the biological active substance OJEF affects the up-regulationand down-regulation of cell signaling pathways that are directly related to cancer development, suppresses cancer cell proliferation,and regulates the cell cycle[3]. After the initiation of apoptosis,phosphatidylserine is exposed on the cellular membrane, which inhibits anti-apoptotic factors such as bcl-2 and activates proapoptotic factors such as bax as caspases. Furthermore, in the nucleus, the activation of endonuclease leads to DNA fragmentation,chromatin condensation, destruction of the nuclear membrane,and blebbing of the nucleus. In cancer, there is an imbalance between cell proliferation and death that results in the continued multiplication of cells. Therefore, control of the cell cycle is a pivotal factor in the study of anti-cancer activity[15,20]. In addition,other studies published to date on whole plants, soluble extracts,and bioactive substances of O. japonicus have solely focused on identifying potential active matters and their anti-bacterial, antioxidative, and anti-inflammatory properties[15-23]. The findings in this study have not been previously reported. Thus, we concentrated on demonstrating the anti-cancer effect of OJEF on HT-29 human colon cancer cells as key regulators affecting or guiding upstream and downstream signaling pathways to regulate apoptosis and cell cycle. To evaluate the effect of OJEF on induction of apoptosis,we conducted flow cytometry analysis with annexin V/PI staining,confocal microscopy with DAPI nuclear staining, and cell cycle analysis. As shown in this study, the total apoptosis rate (31.13%)in the HT-29 cells treated with the 100 μL/mL OJEF was higher than that of the control (8.44%). In addition, the OJEF increased the cell population of sub-G1 phase and S phase in a concentrationdependent manner, implying effective cell cycle arrest in HT-29 cells. When apoptosis in a cell is initiated, first, phosphatidylserine is exposed on the outer cellular membrane, the anti-apoptotic protein bcl-2 is inhibited, and the level of the pro-apoptotic protein bax increases. As a result, cytochrome c leaves from mitochondria,which causes the activation of a cascade of caspases, pro-apoptosis proteins. We demonstrated that OJEF lowered the bcl-2 level and elevated the bax level in a concentration-dependent manner.Apoptosis is induced by various cell death signals and followed by decision making and execution, which are timely regulated by different proteins. The two well-known mechanisms are the death receptor-mediated extrinsic apoptosis pathway and the mitochondriamediated intrinsic apoptotic pathway[8-12]. In the extrinsic pathway,a Fas ligand, a pro-apoptotic death ligand, binds to the Fas receptor,the corresponding death receptor, which then binds to caspase-8 with the assistance of adaptor molecules. When a death inducing signaling complex consisting of a death receptor and caspase-8 is formed, caspase-8 proteins cleave each other to self-activate themselves. As a result, inactive pro-caspase-8 is converted into its active form, cleaved caspase-8[11,12]. In the mitochondria-mediated apoptosis pathway, following cellular DNA damage by extracellular stress, the bax gene, a mitochondrial pro-apoptosis gene, is activated,which induces the outflow of cytochrome c. To the contrary, the anti-apoptotic factor bcl-2 prevents cytochrome c from moving into the cytoplasm to protect the cell against apoptosis. However, when bcl-2 is inhibited by pro-apoptotic proteins, the released cytochrome c activates the mitochondria-mediated intrinsic apoptosis pathway.The released cytochrome c binds caspase recruitment domain and caspase-9 to make an apoptosome. The apoptosome allows nearby pro-caspase-9 molecules to cleave each other to be activated.Cleaved caspase-9 activates the executor caspase-3 to complete the apoptosis process[8-10]. In the present study, OJEF lowered the level of pro-caspase-3, pro-caspase-8 and pro-caspase-9 and raised the expressed amount of cleaved caspase-3, cleaved caspase-8 and cleaved caspase-9 produced in a concentration-dependent manner.When a cell is exposed to extracellular stress, it activates a survival reaction such as proliferation, division, or apoptosis, depending on the type of stress. MAPK compose a crucial cell signaling system to transmit signals from outside the cell into its nucleus. When a cell detects stress signals, it phosphorylates MAPK family members to activate them, and activated ERK, JNK, and p38 then sequentially play pivotal parts in cell proliferation, cycle progression, survival,differentiation, inflammation, apoptosis, and phosphorylation of transcription factors[18,24-26].

Table 2 Quantification of downstream apoptosis-related protein expression levels in HT-29 cells treated with ethylacetate fraction from O. japonicus.

The results are shown as the mean±SD. Ratio of protein levels was observed by Image J program. The density of bands between the target protein and GAPDH was calculated. Cells treated only with 0.1% DMSO was used as control. GAPDH was used as a housekeeping gene. *P＜0.05 was regarded statistically meaningful (vs. control).

Table 3 Quantification of upstream apoptosis-related protein expression levels in HT-29 cells treated with ethylacetate fraction from O. japonicus.

The results are shown as the mean±SD. Ratio of protein levels was observed by Image J program. The density of bands between the target protein and GAPDH was calculated. Cells treated only with 0.1% DMSO was used as control. GAPDH was used as a housekeeping gene. *P＜0.05 was regarded statistically meaningful (vs. control). ERK1/2： extracellular signal regulated kinase 1/2; JNK： Jun-N-terminal kinase.

In this study, it is more likely that anti-cancer property of OJEF is controlled by the mitochondria-mediated pathway regulated by bcl-2 family proteins, ending with the cleavage of pro-caspase-3 to execute programmed cell death by chromatin condensation and formation of apoptotic bodies. In addition, OJEF sends stress signals to HT-29 colon cancer cells, resulting in the activation of MAPK family proteins and the induction of a cascade representing an anti-cancer mechanism through the phosphorylation of ERK, JNK, and p38.

The HT-29 cells treated with OJEF for 12 h were stained with annexin V-PI to investigate the rates of cell apoptosis, and the apoptosis rates for the OJEF untreated control were (3.22±1.93)%for early apoptosis and (5.22±5.03)% for late apoptosis.However, the rates for the group treated with 100 μg/mL OJEF were (14.15±5.01)% for early apoptosis and (16.98±2.82)% for late apoptosis. Besides, the respective rates of early apoptosis treated with different concentrations of OJEF [0 (0.1% DMSO,control), 25, 50, 75, and 100 μg/mL] for 12 h were (3.22±1.93)%,(4.50±2.36)%, (6.48±0.39)%, (8.56±2.12)% and (14.15±5.01)%.The respective rates of late apoptosis were (5.22±5.03)%,(7.07±8.51)%, (9.08±7.62)%, (12.90±1.93)% and (16.98±2.82)%.These results showed elevated levels of early and late apoptosis in the OJEF-treated group in a concentration-dependent manner compared to the control group. Based on the result of the DAPI nuclear staining, which was performed to observe the apoptotic rate,notable morphological changes in the nucleus, such as apoptotic body formation, were observed by the naked eye; these changes in the OJEF-treated group were found to occur in a concentrationdependent manner (Figure 1).

The results from this study demonstrate that O. japonicus extracts contain biological active components that have an anti-cancer effect and provide molecular biology-based data regarding the effects on the target genes at the protein level. We anticipate this plant being developed into a bio-health substance to provide benefits to patients being treated for cancer.

2.2 3组患者的月经改善情况比较 轻度、中度及重度粘连月经改善分别为44例(88.0%)、43例(79.6%)及39例(68.4%)，组间比较差异存在统计学意义(P<0.05)，轻度组改善最明显(P<0.05)，重度组改善最差(P<0.05)。见表2。

Acknowledgments

All authors declare that there are no conflicts of interest.

This work was supported by the 2016 Inje University research grant.

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