A Triterpenoid from Thalictrum fortunei Induces Apoptosis in BEL-7402 Cells Through the P53-Induced Apoptosis Pathway

Thalictrum fortunei S. Moore, a perennial plant distributed in the southeastern part of China, has been used in Traditional Chinese Medicine for thousands of years for its antitumor, antibacterial and immunoregulatory effects. In order to investigate the active components and the mechanism of the anti-tumor effects of Thalictrum fortunei, the growth inhibitory effects of eight triterpenoids isolated from the aerial parts of the plant on tumor cell lines were examined by 3-(4,5)-dimethylthiazoy1-3,5-diphenyltetrazolium bromide (MTT) assay. The MTT-assay results showed that the inhibitory activity of 3-O-β-d-glucopyranosyl-(1→4)-β-d-fucopyranosyl(22S,24Z)-cycloart-24-en-3β,22,26-triol 26-O-β-d-glucopyranoside (1) was stronger than that of the other seven tested triterpenoids on human hepatoma Bel-7402 cell line (Bel-7402), human colon lovo cells (LoVo), human non-small cells lung cancer NCIH-460 cells (NCIH-460) and human gastric carcinoma SGC-7901 cells (SGC-7901) after 48 h treatment in vitro, with the IC50 values of 66.4, 84.8, 73.5, 89.6 μM, respectively. Moreover, the antitumor mechanism of compound 1 on Bel-7402 cell was explored through nucleus dyeing, fluorescence assay, flow cytometry and western blot. The flow cytometric analysis results revealed that compound 1 caused apoptosis and mitochondrial membrane potential (MMP) loss in Bel-7402 cells. A fluorescence assay indicated that intracellular reactive oxygen species (ROS) were markedly provoked by compound 1 treatment compared to control cells. Immunoblot results showed that compound 1 significantly increased the expression levels of cleaved caspase-3, P53 and Bax protein, and decreased the expression level of Bcl-2 protein. These findings indicate that compound 1 inhibits the growth activity of tumor cells, probably through the P53 protein-induced apoptosis pathway.


Introduction
Hepatocellular carcinoma is the eighth most common cancer over the World by number of cases and the third most common cause of death from cancer due to its very poor prognosis. Eighty-two percent of cases are in developing countries, with fifty-five percent being in China [1]. Up to now, chemotherapy has provided significant survival benefits for patients with hepatocellular carcinoma [2], however, it is associated with significant normal tissue toxicity. Thus, it is necessary to find new effective medications for hepatocellular carcinoma. Development of pharmacologically effective agents with little toxicity or few side effects from natural products has become a new important research topic in this area.
Previous reports have indicated that antitumor activity was found in the extract of Thalictrum fortunei [3]. Additionally, many triterpenoids from Thalictrum fortune were isolated and identified [4,5]. Therefore, the aim of this study was to evaluate the cytotoxicity of eight triterpenoids from Thalictrum fortunei on SGC-7901 cells, Bel-7402 cell, LoVo cells and NCIH-460 cells. Further, a representative compound was chose to investigate its anti-tumor mechanism on BEL-7402 cells [6]. To study of the mechanism of cytotoxicity, cell nucleus morphology was observed using Hoechst 33258 staining, and apoptosis were analyzed by flow cytometry. To further elucidate the apoptosis pathway, the mitochondrial membrane potential (MMP), reactive oxygen species (ROS), caspase-3, P53, Bcl-2 and Bax protein expression were also assayed.  Figure 1), by extensive NMR methods, HRESIMS, and hydrolysis respectively. Their purities were all proven to be higher than 98% by HPLC analysis.

Growth Inhibitory Effect of Eight Triterpenoids on SGC-7901, Bel-7402, Lovo and NCIH-460 Cells
The MTT assay was used to determine the cell viability of HSGC-7901, Bel-7402, lovo and NCIH-460 cells after treatment with the different triterpenoids. As shown in Table 1, compound 1 exhibited the strongest in vitro suppressive effect on all the above tumor cells at 48 h after treatment, with IC 50 values of 66.4, 84.8, 73.5, 89.6 μM, respectively, while compounds 2-4 showed milder cytotoxic effects than compound 1 and compounds 5-8 displayed the lower cytotoxic effects among the eight triterpenoids on these four tumor cell lines.

Effect of Compound 1 on the Viability of Bel-7402 Cells
Human hepatoma BEL-7402 cell line was selected as the model strain for cytotoxicity assessment [7][8][9], and to elucidate the mechanisms of the cytotoxicity because it showed the strongest cytotoxity than other compounds in this cell line, as shown in Table 1. We evaluated the inhibitory effect of compound 1 on Bel-7402 cells by the MTT assay. The inhibition ratio of compound 1 in tumor cells at different concentrations (20,40, 80, 160 μM) for 24 h, 48 h and 72 h is shown in Figure 2. Compound 1 exhibited a dose and time-dependent suppressive effect on the growth of Bel-7402 cells. Hence, the possible mechanism of the anti-tumor effects of the triterpenoid on other tumor cells also become an essential objective of our research.

Effect of Compound 1 on the Morphological Changes of Bel-7402 Cells
Hoechst 33258 staining was applied to investigate whether Bel-7402 cells underwent cell death via apoptosis or necrosis. As shown in Figure 3, after administration of different concentrations of compound 1, the Bel-7402 cells revealed marked nuclear condensation, membrane blebbing, nuclear fragmentation and apoptotic bodies, all of which are characteristic of apoptotic programmed cell death. After treatment with compound 1 at the concentrations of 20, 40, 80 μM for 48 h, Hoechst-postive cells were increased from 13.4% to 32.5% and 58.7%, respectively. In contrast, those characteristics were not displayed in control cells.

Flow Cytometry Analysis of Compound 1-Induced Bel-7402 Apoptosis
Annexin V/PI staining assay was used to further confirm the apoptosis induced by compound 1. As shown in Figure 4, after treated with compound 1 at the concentrations of 20, 40, 80 μM for 48 h, the early and median apoptotic cells (right low section of fluorocytogram) were increased strikingly (from 19.3% to 29.6% and 53.4%, respectively) and the late apoptotic and necrotic cells (right upper section of fluorocytogram) were also changed (from 6.3% to 6.4% and 24.6%). These results suggested that apoptosis induction by compound 1 was involved in its antitumor effects.

Effect of Compound 1 on MMP and ROS Levels
To investigate the alteration of mitochondrial membrane potential (MMP) in Bel-7402 cells treated with compound 1, the fluorescent probe JC-1 was used to determine this parameter. As shown in c d b a Figure 5, treatment with different concentrations of compound 1 (at 20, 40, 80 μM) for 48 h induced a significant loss of MMP from 78.8% to 50.3% and 18.6% in a dose-dependent manner. Moreover, to assess the effect of compound 1 on intracellular ROS, after Bel-7402 cells were incubated with concentrations of compound 1 (at 20, 40, 80 μM) for 48 h, intracellular ROS content was significantly increased from 130.45% to 237.3% and 362.7% ( Figure 6) in a dose-dependent manner. Mitochondria are considered to play an important role in apoptosis, the the mitochondrial apoptosis pathway has been described as an important signaling pathway of apoptosis [10,11]. Mitochondrial membrane potential change constitutes an early event in the apoptotic process, which leads to the release of apoptosomes from mitochondria. Further, the apoptosome triggers formation of apoptotic complex and activation of the caspase-9, and leads to the proteolytic activation of caspase-3, the primary caspase of the cell [12].
Loss in MMP induced superabundant ROS, resulting in membrane lipid peroxidation, nitration of proteins, and degradation of DNA, all of which are associated with the course of apoptosis [13]. ROS itself present at high levels also could cause severe damage to DNA, proteins and lipids, which may lead to apoptosis [14]. Our studies indicated that treatment with compound 1 induced increased ROS levels and loss in MMP, both of which promote apoptosis through cytochrome c release from mitochondria to the cytosol.

Effect of Compound 1 on the Expression of Caspase-3, P53, Bcl-2 and Bax Protein
As shown in Figure 7, after incubation with compound 1 at the concentration of 20, 40 and 80 μM for 48 h, the expression of caspase-3 and P53 protein increased dramatically. Furthermore, higher dosage of compound 1 on Bel-7402 cells for 48 h resulted in the decreased Bcl-2 protein levels and the increased Bax protein expression, which leads to a decrease of the Bcl-2/Bax ratio.
These cumulative findings confirmed that p53 was involved in several critical pathways including cell cycle arrest, apoptosis, DNA repair, and cellular senescence, which are essential for normal cellular homeostasis and for maintaining genomic integrity [15]. One pathway of apoptosis is that p53 itself translocates to mitochondria, which are considered to play a important role in apoptosis and where it activates the mitochondrial apoptotic pathway by binding to the anti-apoptotic proteins Bcl-2 and Bcl-xL [16]. Another apoptosis pathway, an accumulation of p53 in the cytosol can act in a similar way to BH3-only proteins, thus inducing the oligomerization of Bax [17]. Finally, p53 leads to the expression of pro-apoptotic proteins, including members of the BH3-only group of Bcl2-related proteins such as Bid, which can trigger the mitochondrial apoptotic pathway [18]. The p53 protein induces the transcription of the TIGAR gene, which lowers the intracellular concentrations of fructose-2,6-bisphosphate and thus decreases overall levels of intracellular reactive oxygen species (ROS) [19]. Our result implied that treatment with compound 1 increased the expression of P53 protein, which leading to cell apoptosis through several pathways such as MMP, ROS, Bax and Bcl-2 protein, etc.
Caspases are a family of intracellular protein involved in the initiation and execution of apoptosis [20]. Activation of caspase-3 was also confirmed by Western blot. As shown in Figure 8, the protein abundance of caspase-3 (32 kDa) increased in a dose dependent manner. The cleavage product (17 kDa) of caspase-3 was also enhanced in Bel-7402 cells after exposure to compound 1. The control cells did not show any evidence of increased caspase-3-like activity and the 17 kDa cleavage product, suggesting caspase-3 was specifically activated in Bel-7402 cells after treatment with compound 1. The results were consistent with the expression levels of antiapoptotic and proapoptotic Bcl-2 family proteins.

Isolation and Identification of Compounds from Thalictrum fortunei S. Moore. Rhizome
The dried aerial parts (5.2 kg) of Thalictrum fortunei were extracted with 95% EtOH (3 × 20 L) under reflux. The EtOH extract was suspended in water and then successively extracted with petroleum ether, EtOAc, and n-BuOH. The n-BuOH solution was concentrated and given a residue (207 g), which was subjected to repeated chromatography on silica gel column and eluted with chloroform-methanol gradient solvent system, then purified with reversed-phase CC and HPLC. The structure of compound was confirmed by spectrum methods (UV, IR, MS, 1 H-NMR and 13 C-NMR).

Cell Lines and Cell Culture
SGC-7901 cells, Bel-7402 Cells, human colon LoVo cells and NCIH-460 cells were obtained from the Animal Experimental Center of Medical College of Sun Yat-Sen University (Guangzhou, China), maintained in RPMI 1640 culture medium plus 10% calf serum and 100 U/mL penicillin, and 100 μg/mL streptomycin in a 37 °C incubator supplied with 95% room air and 5% CO 2 . After 60-80% confluency, the cells were trypsinized with 0.25% trypsin (Amresco, dissolved in PBS, pH 7.4), counted and placed down at needed density for treatment. All cells were prepared to use at 37 °C for 24 hours CO 2 incubation after sub-cultured into new dishes.

Assessment of Anti-Tumor Activities of Eight Triterpenoids from Thalictrum fortunei by MTT Assay
The MTT assay was used to determine the effect of the eight triterpenoids on overall proliferation of SGC-7901, Bel-7402, lovo and NCIH-460 cells. The activity was measured by the previously described method [21]. Briefly, cells in exponential growth were plated in a 96-well plate and grown to 70-80% confluency, followed treatment with the eight triterpenoids at different concentrations (5, 10, 20, 40, 80, 160 μM) for 48 h, respectively. Then, MTT (100 μL, 5 mg/mL) in PBS was added, and the cells were further incubated at 37 °C for 4 h to allow for complete cleavage of the tetrazolium salt by metabolically active cells. Finally, the supernatant was discarded and dimethyl sulphoxide (DMSO, 150 μL) was added. The 96-well plate was vibrated on a micro-vibrator for an additional 10 min, and the optical density of each well was measured at λ 490nm with an enzyme-immunoassay instrument (Thermo Multiskan Ascent). The growth inhibitory ratio of each triterpenoid was calculated based on the following formula:

Assessment of Anti-Tumor Activity of Compound 1 on Bel-7402 Cells by MTT Assay
Base on the above MTT-colorimetric assay, the Bel-7402 cells were treated with compound 1 (20,40, 80, 160 μM) for 24 h, 48 h and 72 h, respectively. MTT (5 mg/mL) in PBS was then added to wells and the cells incubated at 37 °C for 4 h to allow for complete cleavage of the tetrazolium salt by metabolically active cells. Finally, the supernatant was removed and DMSO (150 μL) was added. The 96-well plate was vibrated on micro-vibrator for additional 10 min, and the optical density of each well was measured at λ 490nm with the enzyme-immunoassay instrument (Thermo Multiskan Ascent).

Apoptosis Analysis by Hoechst 33258 Staining
Hoechst 33258 staining was carried out as previously described [22]. Briefly, the Bel-7402 cells in logarithmic growth phase were placed down at a final concentration of 1 × 10 5 cells/well in a 6-well culture plate. A slide was placed on the bottom of each well to allow cells to grow on its surface as a monolayer. After confluence, the cells were exposed to chrysin (20, 40, 80 μM) for 48 h. For Hoechst 33258 staining, the cells were fixed with ice-cold methanol at room temperature for 5 min, washing twice with ice-cold PBS, and then loaded with Hoechst 33258 for additional 20 min. The changes in the nuclei of cells after Hoechst 33258 staining were observed under a fluorescence microscope (Olympus, BX-60 Japan) at 200× magnification for 10 min.

Determination of Cell Apoptosis by Annexin V/PI Double Staining
The Bel-7402 cells were exposed to compound 1 (20,

Measurement of MMP by Flow Cytometry
MMP was detected as described in a previous report [23]. Briefly, after cells were pre-incubated with compound 1 at the different concentration, they were centrifuged for 5 min at 2,000 rpm, and collected at the concentration of 5 × 10 5 , and incubated in Hank's solution containing 10 μg/mL JC-1 for 30 min at 37 °C, then was centrifuged for 5 min at 2,000 rpm. Finally, cells sample was collected and washed twice with pre-warmed Hank's solution (37 °C) followed by re-floating and analyzing with flow cytometry with an excitation wavelength of 480 nm and an emission wavelength of 530 nm.

Measurement of Intracellular ROS by Fluorescent Methods
Intracellular ROS was monitored by using the DCFH-DA fluorescent probe [24]. Intracellular H 2 O 2 or low-molecular-weight peroxides can oxidize DCFH-DA to the highly fluorescent compound dichlorofluorescein (DCF), Bel-7402 cells were harvested after 48 h treatment by compound 1 (20, 40, 80 μM) and disrupted in cell lysis buffer. Cells were incubated with 10 mM DCFH-DA at 37 °C for 30 min, and then washed twice with PBS. Finally the fluorescence intensity of DCF was measured in the enzyme-immunoassay instrument (Thermo Multiskan Ascent) with an excitation wavelength of 485 nm and an emission wavelength of 535 nm.

Western Blot Analysis
Bel-7402 cells were harvested after 48 h treatment by compound 1 (20, 40, 80 μM) and disrupted in cell lysis buffer, and then they were centrifuged at 13,000 g for 10 min. The supernatant was collected and used for further analyses. To analyze P53, caspase-3, Bax and Bcl-2 proteins, the cytosolic fractions were prepared by differential centrifugation using the method described by Sugawara [22]. The protein concentration was determined using the Bradford staining method, after which equal amounts of protein (20 μg) were electrophoresedon 10-15% density SDS-acrylamide gels. Following electrophoresis, the proteins were transferred from the gel to a nitrocellulose membrane using an electric transfer system. Non-specific binding was blocked with 5% skim milk in TBST buffer (5 mM Tris-HCl, pH 7.6, 136 mM NaCl and 0.1% Tween-20) for 1 h. The blots were incubated with antibodies against caspase-3 (1:250), P53 (1:500), Bcl-2 (1:500), Bax (1:1,000), and β-actin (1:500) overnight at 4 °C, after which they were washed three times with 1× TBST. The blots were incubated for 1 h at room temperature with a 1:5,000 dilution of horseradish peroxidase-labeled anti-rabbito or anti-mouse IgG. They were washed three times with 1× TBST, after which they were developed using the ECL Western detection reagents.

Statistical Analysis
The significance of the differences between the different groups was determined with the Student's t-test. The results are presented as mean ± S.D. of three or six independent experiments. The differences were considered significant at p < 0.05.

Conclusions
In conclusion, our studies demonstrate for the first time that eight triterpenoids isolated from Thalictrum fortunei show different potent growth inhibitory activities in vitro on several tumor cell lines. Compound 1 induces apoptosis by up-regulating the expressions of caspase-3, P53 and Bax proteins and down-regulating the expression of Bcl-2 protein in Bel-7402 cells, which may contribute to the anti-tumor effects of Thalictrum fortunei. These findings provide a relevant basis for developing triterpenoids from Thalictrum fortunei as an lead compound for cancer medical treatment.