Potent effects of dioscin against gastric cancer in vitro and in vivo.
Background: We previously reported the effect of dioscin on human gastric carcinoma SGC-7901 cells, but its effects on other gastric cancers are still unknown.
Purpose: The present paper aimed to demonstrate the activity of dioscin against human gastric carcinoma MGC-803 and MKN-45.
Study design: In our study, MGC-803 and MKN-45 cells were used to examine the effects of dioscin on human gastric carcinoma in vitro. The effects of dioscin against human gastric carcinoma in vivo were accomplished by the xenografts of MGC-803 cells in BALB/c nude mice.
Methods: AO/EB and DAPI staining, TEM, single cell gel electrophoresis and flow cytometry assays were used in cell experiments. Then, an iTRAQ-based proteomics approach, DNA and siRNA transfection experiments were carried out for mechanism investigation.
Results: In MGC-803 cells, dioscin caused DNA damage and mitochondrial change, induced ROS generation, [Ca.sup.2+] release and cell apoptosis, and blocked cell cycle at S phase. In vivo results showed that dioscin significantly suppressed the tumor growth of MGC-803 cell xenografts in nude mice, In addition, dioscin markedly inhibited cell migration, caused Cytochrome c release and adjusted mitochondrial signal pathway. Then, an iTRAQ-based proteomics approach was carried out and 121 differentially expressed proteins were found, in which five biomarkers associated with cell cycle, apoptosis and migration were evaluated. Dioscin significantly up-regulated the levels of GALR-2 and RBM-3, and down-regulated CAP-1, Tribbles-2 and CIiC-3. Furthermore, overexpressed DNA transfection of CAP-1 enhanced cell migration and invasion, which was decreased by dioscin. SiRNA to Tribbles-2 affected the protein levels of Bcl-2, Bax and MAPKs, suggesting that dioscin decreased Tribbles-2 level leading to cell apoptosis.
Conclusion: Our works confirmed the activity of dioscin against gastric cancer. In addition, this work also provided that dioscin is a new potent candidate for treating gastric cancer in the future.
Due to the high mortality and low 5-year survival rate, gastric cancer has become the second leading cause of cancer-related deaths worldwide (Schildberg et al. 2012), and almost 990,000 cases are diagnosed annually (Lee et al. 2014). Although some chemical drugs used in clinical chemotherapy are effective, some serious side effects accompany with the therapy. Thus, development of new drugs or candidates with high efficiency and low side effects is important.
Isobaric tags for relative and absolute quantification (iTRAQJ, one widely used proteomics technique, allows the differential labeling of peptides from distinct proteomes, which provides a more comprehensive approach for finding biomarkers (Weist et al. 2008). This technique with great popularity in quantitative proteomic application has been successfully used to identify the differentially expressed proteins in gastric cancers (Hu et al. 2010; Xu et al. 2015).
Dioscin (shown in Fig. 1A), a typical natural product, is derived from some medicinal plants. Pharmacological investigations have shown that dioscin has anti- fungal, anti-virus and hepatoprotective activities (Ikeda et al. 2000; Lu et al. 2011). In addition, more efforts have been made to investigate the anti-cancer activities of dioscin on human LNCaP prostate carcinoma, human lung cancer, human esophageal cancer, human leukemia, etc. (Chen et al. 2014; Lv et al. 2013; Wang et al. 2013b; Wang et al. 2012). In our previous works, dioscin significantly induced the apoptosis of human gastric carcinoma SGC-7901 cells (Hu et al. 2013), but its effects and mechanisms on other gastric cancers are still unknown to the best of our knowledge.
Therefore, the aim of this study was to confirm the effects of dioscin on human gastric carcinoma in vitro and in vivo. Then, an iTRAQ-based proteomics approach was used for the mechanism investigation.
Materials and methods
Chemicals and reagents
Dioscin with the purity of >98% was prepared in our laboratory (Hu et al., 2012; Yin et al., 2010). Cell cycle and apoptosis analysis Kits, and lysis buffer were all purchased from Beyotime Institute of
Biotechnology (Shanghai, China). 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), In situ Cell Death Detection Kit, were obtained from Roche Diagnostics, Mannheim, Germany. Acridine orange (AO) and ethidium bromide (EB) fluorescent dyes were purchased from Nanjing KeyGen Biotech. Co. Ltd., Nanjing, China. 4,6-diamidino-2-phenylindole (DAPI) was provided by Sigma (St. Louis, MO, USA).
The human gastric cancer MGC-803 and MKN-45 cell lines were provided by China Infrastructure of Cell Line Resources (Beijing, China). The cancer cells were cultured in RPMI-1640 medium with low carbohydrates containing 10% fetal bovine serum (FBS) at 37 [degrees]C in 5% C02. In the following experiments, the cells were allowed to adhere in the logarithmic growth phase for 24 h prior to treatment.
Cell viability assay
The MGC-803 and MKN-45 cells were plated in 96-well plates at a density of 1 x [10.sup.5] cells/well and cultured at 37[degrees]C in 5% C[O.sub.2] for 24 h, and then treated with different concentrations of dioscin (1.25, 2.5 and 5.0 [micro]g/ml) under different treatment times (6, 12 and 24 h) for MTT assay. Total of 10 [micro]l MTT solution (5.0mg/ml in PBS) was added, and the cells were incubated at 37 [degrees]C for 1 h, then 150 [micro]l DMSO was added to dissolve the formazan crystals. The absorbance was measured at a wavelength of 570 nm using a microplate reader (Thermo, USA).
AO/EB and DAPI staining
After incubation, the cells were washed by cold PBS, then 20 [micro]l solution containing the same volume of AO (100 [micro]g/ml in PBS) and EB (100 [micro]g/ml in PBS) was added. For DAPI staining, the cells were treated and washed as mentioned above, stained with DAPI (1.0
[micro]g/ml) solution for 10 min at 37[degrees]C, and then washed twice with PBS. The images were obtained using an inverted fluorescence microscope (OLYMPUS, Osaka, Japan).
Transmission electron microscopy (TEM) assay
The MGC-803 cells (2 x [10.sup.5] cells/ml) were plated in 6-well plates, harvested and fixed overnight at 4[degrees]C in 2% glutaraldehyde. After washing for three times (each for 15 min) in 0.1 M sodium cacodylate buffer, the cells were fixed in 1% osmium tetroxide for 2 h. The pretreated samples were used for ultramicrotomy and collected on copper grids, and the obtained sections were then stained and observed using a transmission electron microscope (JEM-2000EX, JEDL, Japan).
Single cell gel electrophoresis assay
After being treated with different concentrations of dioscin (1.25, 2.5 and 5.0 [micro]g/ml) for 24 h, the images were observed by a fluorescence microscope (OLYMPUS, Osaka, Japan) according to the manufacturer's instructions (CELL BIOLABS, INC, USA).
Measurement of cell cycle
The MGC-803 cells incubated in six-well plates were treated by dioscin (1.25, 2.5 and 5 [micro]g/ml for 24 h, or treated by 5.0 [micro]g/ml of dioscin for 6, 12 and 24 h), collected, washed twice by ice-cold PBS, centrifuged at 1000g for 5 min, and fixed with 75% ethanol at 4[degrees]C for 24 h. After that, the cells were stained with PI staining solution for 30 min at 37[degrees]C, and analyzed by flow cytometry (Becton-Dickinson, USA).
Detecting cell apoptosis by flow cytometry assay
Cell apoptosis was analyzed by flow cytometry based on the Annexin V-FITC/ propidium iodide apoptosis kit (Beyotime, Shanghai, China). Briefly, the MGC-803 cells were treated by dioscin as mentioned above, then collected and washed twice with cold PBS. After that, the cells were stained with Annexin V-FITC/propidium iodide and analyzed by flow cytometry assay (Becton-Dickinson, NJ, USA).
Detection of intracellular ROS accumulation and [Ca.sup.2+] release
The MGC-803 cells were plated in 6-well plates at a density of 1 x [10.sup.5] cells/well and treated by dioscin. After treatment, the cells were collected and re-suspended in 500 [micro]l DCFH-DA (10.0 [micro]M) for determining ROS and in 500 [micro]l of Fluo-3/AM (2.5 [micro]M) for detecting [Ca.sup.2+], which were all analyzed by flow cytometry assay (Becton-Dickinson, NJ, USA).
We transduced firefly luciferase genes into MGC-803 cells by using lentivirus vectors of pLEX system (Berthold, Shanghai, China). The cells were seeded in 6-well plates, cultured with RPMI-1640 medium with 10% FBS for 24 h, and transduced by lentivirus infection in RPMI-1640 medium with 50 [micro]g/ml of lentivirus vectors in every single well for 12 h. Then, puromycin selection was performed in the presence of 2 mg/ml puromycin for 2 weeks. The surviving colonies were harvested and cultured independently, and MGC-803-luc cells were obtained. Then, the transduced cells were screened using a bioluminescence, and the MGC-803-Luc cells with the highest luciferase activity were used in vivo study. The BALB/c nude mice weighing 15-20 g in 4-6 week-old were provided by the Experimental Animal Center at Dalian Medical University, Dalian, China (SCXK: 2013-0006). All animals were housed in a controlled environment at 23 [+ or -] 2[degrees]C under a 12-h dark/light cycle with free access to food and water. The animal maintenance and experiments were performed in accordance with the guidelines of the Animal Care and Use Committee. Optimized MGC-803-luc cells (2 x [10.sup.6]) were suspended in 0.2 ml of PBS and injected subcutaneously into the right buttock region of the mice. Three days after implantation, the mice were randomly divided into three groups in which the mice in control group were received by oral 0.5% CMC-Na, and the mice in other two groups were oral administrated with dioscin at the doses of 40 and 80 mg/kg for 30 days. In the process, the side length of the tumor was measured to calculate the tumor volume based on the formula: V=A x [B.sup.2]/2, where A means the lager perpendicular diameters and B is the smaller perpendicular diameters. At the end of the test, the mice were imaged by the luciferase signals emitted from the cells (IVIS 200 Imaging System, Xenogen, MA, USA), then the animals were sacrificed and the tumors were obtained, photographed and weighted.
Labeling with 4-plex iTRAQ reagents
Based on our previous study, iTRAQ assay was carried out (Xu et al. 2014). Briefly, the samples collected from the dioscin-treated (5.0 [micro]g/ml for 24 h) or untreated MGC-803 cells were mixed with lysis buffer including 50 mM Tris-HCl, 8M urea, 4% CHAPS, 2.5 M thiourea and 65 mM DTT. Insoluble debris was pelleted by centrifugation at 100, 000 X g for 60 min. The total proteins were extracted and quantified using the Bradford Assay Kit (BioRad, Hercules, CA, USA). The samples were labeled with iTRAQ reagents as follows: control, iTRAQ reagent 114 and 117; dioscin, iTRAQ reagent 115 and 116 with two biological replicates. The proteins with equal contents in each biological replication were reduced, alkylated, and digested by trypsin according to the protocol provided by the manufacturer (Applied Biosystems, Foster City, CA, USA).
Strong cation exchange chromatography and nano-LC-TOF-MS/MS identification
First, the labeled peptides were fractionated by strong cation exchange chromatography (SCX), and 3 fractionations were collected and dried in the present work. Further separation was achieved by nano-LC-MS/MS. The peptides were separated on a polysulfoethyl column (100 x 4.6 mm, i.d., 5 [micro]m, 200 [Angstrom] pore size) using an AKTA Purifier 100 unit (GE Healthcare, Niskayuna, NY, USA) system. The mobile phase consisted of buffer A and buffer B (10 mM K[H.sub.2]P[O.sub.4], 500 mM KCl and 25% ACN, pH 3.0) was set at a flow rate of 1 ml/min with gradient elution. Then, a second dimension separation was performed on a nanoscale HPLC system (EASY-nLC from Proxeon Biosystems, Odense, Denmark) coupled with an Orbitrap Q Exactive (Thermo Fisher Scientific, MA, USA) for online mass spectrometric (MS) analysis. Peptide was captured on an EASY [C.sub.18] column (Thermo Scientific, MA, USA). Mobile phase consisted of solution A (0.1% aqueous formic acid) and solution B (0.1% formic acid acetonitrile) with gradient separation as follows: 0-5% solution B for 2 min; 5-35% solution B for 120 min; 35-80% solution B for 15 min; 80% solution B for 15 min. Each sample was analyzed in parallel three times.
Database search and iTRAQ quantification
Protein identification and quantification were performed with MaxQuant software suite (http://www.ebi.ac.uk/IPI/IPI human, html) version 1.3.O.5. The peak list files were searched for protein identification using MaxQuant against the International Protein Index (IPI) human database (ipi. human 3.17.fasta). We accepted only peptides and proteins with a false discovery rate (FDR) of less than 1%. The relative protein expression in treatment groups was calculated in relation to the untreatment. Finally, the expression was modified (fold change > 3.0-fold) with p-value < 0.05 were selected as the differentially expressed proteins.
The injury line was created in confluent cells and washed with PBS, then the MGC-803 cells were treated with dioscin (0.3, 0.6 and 1.2 [micro]g/ml) under different treatment times (6, 12 and 24 h). After that, the cells were washed, and the images were photographed by the fluorescence microscope (OLYMPUS, Japan).
Transwell invasion assay
The MGC-803 cells (3 x [10.sup.4] cells/well for invasion assay or 8 x [10.sup.4] cells/well for migration assay) were added to the upper chamber with 200 [micro]l culture medium with dioscin (0.3, 0.6 and 1.2
[micro]g/ml), and the lower chamber was filled with 500 [micro]l medium containing 10% FBS. After 6, 12 and 24 h treatment at 37[degrees]C, the surface of the transwell filter was swept by cotton swabs. The cells were counted under a microscope in five random fields.
Detection of Cytochrome c release
The cells were incubated with the primary antibody overnight at 4[degrees]C. On the second day, the plates were washed twice with PBS, incubated with the secondary antibody for 1 h at 37[degrees]C, washed by PBS and dyed with DAPI (5.0 [micro]g/ml) for 5 min. The images of the cells were obtained by a laser scanning confocal microscope (Leica, TCS SP5, Germany).
Western blotting assay
The lysates from MGC-803 cells in different groups were extracted and centrifuged at 15,000 g for 10 min at 4[degrees]C, then the total proteins were obtained. An aliquot (50 [micro]g protein) was loaded onto a 8%-12% SDS-PAGE gels and separated electrophoretically. Then the target proteins were transferred to a PVDF membrane (Millipore, USA). After blocking the PVDF membrane in 5% dried skim milk (Boster Biological Technology, China) for 3 h at room temperature, the membrane was incubated overnight at 4[degrees]C with primary antibodies (Supplementary Table 1) for 3 h at room temperature. Protein detection was performed based on an enhanced chemiluminescence (ECL) method and photographed by using a BioSpectrum Gel Imaging System (HR410, UVP, USA). In order to eliminate the variations, data were adjusted to GAPDH expression: IOD of objective protein versus IOD of GAPDF1 expression.
CAP-1 overexpression DNA transfection experiment
CAP-1-targeted DNA overexpression and control DNA were transfected into MGC-803 cells using Lipofectamine 2000 reagent, (DNA: Lipofectamine 2000=1 [micro]g: 2.5 [micro]l). The cells were used for other experiments after transfection, and the protein level of CAP-1 was detected.
Tribbles-2 siRNA transfection experiment
Briefly, Tribbles-2-targeted siRNA and control siRNA were dissolved separately in OptiMEM. The solutions were equilibrated for 5 min at room temperature, and each RNA solution was combined with Lipofectamine 2000 reagent (siRNA: Lipofectamine 2000=1 pmol: 0.05 [micro]l). The samples were mixed gently and allowed to form siRNA liposomes for 20 min. The MGC-803 cells were transfected with the transfection mixture in antibiotic-free cell culture medium, and the protein levels of Tribbles-2, Bcl-2, Bax and MAPKs were detected.
All values for each group were given as mean and standard deviation (SD). The data were analyzed by one-way analysis of variance (ANOVA) coupled with LSD in Post Hoc Multiple Comparisons using the SPSS Statistics 18.0 (IBM, New York, USA). Differences were considered significant when p < 0.05 or 0.01.
Dioscin inhibits cell viability and induces morphological changes
The MTT results showed that the proliferation of MGC-803 and MKN-45 cancer cells was significantly inhibited by dioscin with time- and dose-dependent manners (Fig. 1B). The inhibiting ratio of MGC-803 cells treated by 5.0 [micro]g/ml of dioscin for 24 h was reached to 56.37%, and the ratio of MKN-45 cells treated by 5.0 [micro]g/ml of dioscin for 48 h was reached to 51.64%. Thus, MGC-803 cells were selected for subsequent experiments. As shown in Fig. 1C, the apoptotic and necrotic cells with orange fluorescence were gradually increased with the increased concentration of dioscin and treatment time. Through DAPI staining, the nucleus of the cells were condensed and the nuclear apoptotic bodies were formed treated by dioscin. These results indicated that dioscin strongly inhibited the proliferation of human gastric cancer cells.
Dioscin induces ultrastructure changes and DNA damage
Under TEM investigation, the cells in control group exhibited normal ultrastructure including round nuclei, sharp edges and complete nuclear membrane. While the cells treated by dioscin (5.0 [micro]g/ml for 24 h) displayed the typical features of apoptosis including nuclear chromatin condensation and marginalization (Fig. 1D). As shown in Fig. 1E, the cell shapes were round and DNA remained in nuclear matrix in control group. With the noticeable decrease of DNA content in head and appearance of comet, DNA damage caused by dioscin were apparently observed in SCGE assay.
Dioscin affects cell cycle arrest and induces cell apoptosis
As shown in Fig. 2A, the cells at G0/G1 phase were decreased, whereas the cells at S phase were increased with the increased dose of dioscin. In Fig. 2B, the percentages of the apoptotic cells were significantly increased from 4.49 [+ or -] 5.19% to 57.17 [+ or -] 14.71% treated by dioscin (1.25, 2.5 and 5.0 [micro]g/ml) for 24 h, and also significantly increased from 8.33 [+ or -] 1.39% to 61.64 [+ or -] 4.22% treated by 5.0 [micro]g/ml of dioscin for 6, 12 and 24 h. These results demonstrated that dioscin caused cell cycle arrest at S phase and induced apoptosis of MGC-803 cells.
Dioscin affects ROS generation and [Ca.sup.2+] release
As shown in Fig. 2C, treatment of MGC-803 cells with dioscin (2.5 and 5.0 [micro]g/ml) markedly caused ROS generation (p<0.01) compared with control cells. In addition, the cell concentration of [Ca.sup.2+] was also significantly increased (p<0.01) treated by 5.0 [micro]g/ml of dioscin (Fig. 2D).
Dioscin inhibits tumor growth of MGC-803-luciferase cells in nude mice
As shown in Fig. 3A, at the beginning of the eighth day, the tumor volume in control group was larger than those of in dioscin-treated groups with p < 0.01, and the significant difference was also found based on bioluminescence imaging (Fig. 3B). At the end of the process, dioscin-treated groups showed a significant decrease in tumor weight (Fig. 3C-D) compared with control group (p<0.01).
Different expressed proteins from iTRAQ-based proteomics
A total of 691 proteins were identified, and 121 differentially expressed proteins (fold change > 3.0-fold with p-value <0.05) were found (Supplementary Table 2) from MGC-803 cells caused by dioscin. Five differentially expressed proteins including chloride intracellular channel 3 (CLiC-3), RNA-binding motif protein 3 (RBM-3), galanin receptor protein 2 (GALR-2), adenylate cyclase-associated protein 1 (CAP-1) and Tribbles-2 associated with cell cycle, apoptosis and migration were further validated. As shown in Fig. 4A, compared with control group, dioscin markedly up regulated the protein levels of RBM-3, GALR-2, and down-regulated the protein levels of CAP-1, Tribbles-2 and CliC-3.
Dioscin inhibits the migration and invasion of MGC-803 cells
As shown in Fig. 4B, the cell numbers transferred to the scratch in dioscin-treated groups were significantly less than those of in control group. As shown in Fig. 4C, the crystal violet staining showed that dioscin significantly decreased the cell numbers passing through the polycarbonate membranes.
Dioscin adjusts mitochondrial and MAPK signaling pathways
As shown in Fig. 5A, the cells in control group showed a point or massive staining pattern, while the staining of dioscin-treated (5.0 [micro]g/ml) cells was diffuse, suggesting that dioscin caused the release of Cytochrome c from mitochondria into cytosol. As shown in Fig. 5B, dioscin significantly down-regulated the protein level of Bcl-2, and up-regulated the protein levels of Bax, caspase-3 and -9. In addition, the levels of MAPKs phosphorylation were markedly up-regulated by dioscin compared with control group.
Dioscin affects cell migration after transfecting over-expressed CAP-1
The expression of CAP-1 DNA plus dioscin group was lower than that of in CAP-1 DNA treatment group with p < 0.01 (Fig. 6A). In wound-healing assay, the cell numbers transfer to the scratch in CAP-1 DNA plus dioscin treatment group were significantly less than those of CAP-1 DNA group, suggesting that dioscin affected the wound-healing capacity of MGC-803 cells. In transwell assay, the crystal violet staining showed that CAP-1 DNA plus dioscin treatment group showed less cell numbers passing through the polycarbonate membranes compared with CAP-1 DNA group. As shown in Fig. 6B-C, dioscin effectively inhibited cell migration after transfective overexpression CAP-1 DNA in MGC-803 cells.
Dioscin affects MGC-803 cells after transfecting Tribbles-2 siRNA
As shown in Fig. 6D, based on the small interfering RNA (siRNA) transfection of Tribbles-2, the expression levels of Tribbles-2 and Bcl-2 in siRNA group were significantly decreased compared with control group, but the expression levels of Bax, p-JNK/JNK, pERK/ERK and p-p38/p38 were all increased. In Tribbles-2 siRNA plus dioscin group, the expression levels of Tribbles-2 and Bcl-2 were also significantly decreased, but the levels of Bax, p-JNK/JNK, p-ERK/ERK and p-p38/p38 were all increased. Furthermore, dioscin significantly increased the apoptotic cells based on flow cytometry assay (Fig. 6E) Combination of Tribbles-2 siRNA and dioscin slightly altered the effect of Tribbles-2 siRNA with no statistically significant (p > 0.05).
Our previous study confirmed that dioscin can induce the apoptosis of human SGC-7901 cells at the doses of 0.65, 1.3 and 2.6 [micro] g/ml through death receptor and mitochondrial pathways. In the present work, dioscin at the doses of 1.25, 2.5 and 5.0 [micro] g/ml caused DNA damage, mitochondrial changes, induced ROS generation, [Ca.sup.2+] release, cell apoptosis, and blocked cell cycle at S phase in MGC-803 cells. In vivo results showed that dioscin significantly suppressed the tumor growth of MGC-803 cell xenografts in nude mice. In addition, diocsin significantly inhibited cell migration, caused Cytochrome c release and adjusted mitochondrial signal pathway. These results indicated that dioscin showed cytotoxic effects to SGC-7901 and MGC-803 cancer cells through adjusting apoptosis and mitochondrial pathways. In order to synthetically evaluate the effect of dioscin against gastric cancers, the cytotoxic effect in poorly differentiated human gastric cancer MKN-45 cell line was tested. In this paper, dioscin displayed more cytotoxic effects in MGC-803 cells instead of MKN-45 cells, suggesting that diocsin possessed a more powerful anti-cancer effect on gastric adenocarcinoma.
It is known that some biological processes including apoptosis, cell cycle arrest and DNA damage associated with the actions of anti-cancer drugs have been reported (Mondal et al. 2014). Massive accumulation of intracellular ROS is regarded as a signal of apoptotic initiation, which is known to trigger a series of mitochondria-associated events, which can cause DNA damage, cell cycle arrest and endoplasmic reticulum stress (Xiao et al. 2015). And endoplasmic reticulum stress can increase [Ca.sup.2+] level and trigger cell apoptosis (Wang et al. 2013a). In the present work, dioscin significantly caused ROS generation, and induced [Ca.sup.2+] release, cell apoptosis, cell cycle arrest and DNA damage, suggesting that the effects of dioscin on MGC-803 cells in vitro may be through affecting ROS and the related biological processes.
In recent decades, growing scientific evidences support the role of ion channels in the development of cancers (Cuddapah and Sontheimer 2011). Both potassium selective pores and chloride permeabilities are considered as the most active channels during tumorigenesis (Jentsch et al. 2002). High rate of proliferation, active migration and invasiveness into non-neoplastic tissues are specific properties of neoplastic transformation. All these actions require partial or total involvement of a new protein family, chloride intracellular channels (CLiCs), which is ubiquitously expressed and also participates in the regulation of cell cycle (Zhang et al. 2013). It is known that CLiC-3 precipitates disassembly of cell matrix adhesion and induces migration (Cuddapah and Sontheimer 2010). In the present paper, our results clearly demonstrated that dioscin significantly down-regulated the expression level of CLiC-3, thereby inhibited cell migration, which should be one potent mechanism of the chemical against gastric cancer.
RNA-binding motif protein 3 (RBM-3), a glycine rich protein containing a RNA- recognition motif (RRM), can bind with DNA and RNA (Boman et al. 2013). Galanin receptor protein 2 (GALR-2), one galanin receptor, has been considered to be one potential tumor suppressor and therapeutic target (Berger et al. 2004). Adenylate cyclase-associated protein 1 (CAP-1), is associated with the stimulation of cellular proliferation, migration, adhesion, extracellular matrix formation, as well as the regulation of angiogenesis and tumorigenesis (Wang et al. 2009). In the present paper, dioscin significantly decreased the expression levels of RBM-3, GALR-2 and CAP-1, suggesting that RBM-3, GALR-2 and CAP-1 maybe the molecular targets of dioscin against gastric cancer.
Mitogen-activating protein kinases (MAPK) activation is also involved in apoptosis. There are three major extracellular signal-regulated kinases involved in MAPK pathways: ERK, c-Jun aminoterminal kinase JNK and p38 kinase. Tribbles-2 can serve as a molecular switch associated with MAPK pathway in response to stimulation. The results in our work obviously demonstrated that dioscin markedly down-regulated the expression of Tribbles-2, and decreased by Tribbles-2 siRNA transfection in MGC-803 cells. In a word, dioscin significantly reduced Tribbles-2 level to induce cell apoptosis and initiate MAPK pathway.
Bcl-2, one of the anti-apoptotic proteins, can protect cells from apoptosis through preventing the formation of mitochondrial pores, protecting membrane integrity, and inhibiting Cytochrome c release (Brenner et al. 2000). Bax, a pro-apoptotic protein, can trigger apoptosis and cause Cytochrome c release. Cytochrome c can activate caspases and stimulate apoptosis (Huttemann et al. 2011). It is reported that MAPK activates Bax, initiates mitochondrial translocation and plays an important role in the regulation of Bcl-2 family (Gogvadze et al. 2006). Our results clearly indicated that dioscin markedly up-regulated the levels of Bax, caspase-3, caspase-9, p-JNK/JNK, p-ERK/ERK, p-p38/p38, and down-regulated Bcl-2 level. In summary, dioscin significantly reduced Tribbles2 level to initiate MAPK pathway and activate the Bcl-2 family, resulting in the apoptosis of MGC-803 cells. These results also showed that affecting mitochondria pathway may be one of the potential mechanisms of dioscin against gastric cancer.
Our results showed that dioscin has potent effects against human MGC-803 gastric cancer in vivo and in vitro via inducing cell apoptosis, DNA damage, mitochondrial structure changes, ROS and [Ca.sup.2+] generation, cell cycle arrest and cell migration. The method of iTRAQ-based proteomics was used for mechanism study and some biomarkers including CAP, Tribbles, RBM, GALR and CLiCs associated with apoptosis, cell migration and cell cycle were found. Our works confirmed the potent effect of dioscin against gastric cancer and provide new insights into the mechanisms of the compound as one new potent candidate for the cancer treatment in the future.
Received 21 December 2015
Revised 24 January 2016
Accepted 26 January 2016
Conflict of interest
The authors declare no competing financial interests.
This work was supported by the Foundation of Innovation Team of Education Ministry (IRT13049) and the Program for Liaoning Innovative Research Team in University (LT2013019).
Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2016.01.012.
Berger, A., Lang, R., Moritz, K., Santic, R., Hermann, A., Sperl, W., Kofier, B., 2004. Galanin receptor subtype GalR2 mediates apoptosis in SH-SY5Y neuroblastoma cells. Endocrinol. 145, 500-507.
Boman, K., Segersten, U., Ahlgren, G., Eberhard, J., Uhlen, M., Jirstrom, K., Malmstrom, P.U., 2013. Decreased expression of RNA-binding motif protein 3 correlates with tumour progression and poor prognosis in urothelial bladder cancer. BMC Urol. 13, 17.
Brenner, C., Cadiou, H., Vieira, H.L., Zamzami, N., Marzo, I., Xie, Z., Leber, B., Andrews, D., Duclohier, H., Reed, J.C., Kroemer, G., 2000. Bcl-2 and Bax regulate the channel activity of the mitochondrial adenine nucleotide translocator. Oncogene 19, 329-336.
Chen, J., Li, H.M., Zhang, X.N., Xiong, C.M., Ruan, J.L, 2014. Dioscin-induced apoptosis of human LNCaP prostate carcinoma cells through activation of caspase -3 and modulation of Bcl-2 protein family. J. Huazhong Univ. Sci. Technol. 34, 125-130.
Cuddapah, VA., Sontheimer, H., 2010. Molecular interaction and functional regulation of C1C-3 by [Ca.sup.2+]/calmodulin-dependent protein kinase II (CaMKII) in human malignant glioma. J. Biol. Chem. 285, 11188-11196.
Cuddapah, V.A., Sontheimer, H., 2011. Ion channels and transporters [corrected] in cancer. 2. Ion channels and the control of cancer cell migration. Am. J. Physiol. Cell Physiol. 301, C541-C549.
Gogvadze, V., Orrenius, S., Zhivotovsky, B., 2006. Multiple pathways of cytochrome c release from mitochondria in apoptosis. Biochim. et Biophys. Acta 1757, 639-647.
Hu, H.D., Ye, F., Zhang, D.Z., Hu. P., Ren, H., Li, S.L., 2010. iTRAQ quantitative analysis of multidrug resistance mechanisms in human gastric cancer cells. J. Biomed. Biotechnol. 2010, 571343.
Hu, M., Xu, L., Yin, L., Qi, Y., Li, H., Xu, Y., Han, X., Peng, J., Wan, X., 2013. Cytotoxicity of dioscin in human gastric carcinoma cells through death receptor and mitochondrial pathways. J. Appl. Toxicol. 33, 712-722.
Huttemann, M., Pecina, P., Rainbolt, M., Sanderson, T.H., Kagan, V.E., Samavati, L, Doan, J.W., Lee, I., 2011. The multiple functions of cytochrome c and their regulation in life and death decisions of the mammalian cell: From respiration to apoptosis. Mitochondrion 11, 369-381.
Ikeda, T., Ando, J., Miyazono, A., Zhu, X.H., Tsumagari, H., Nohara, T., Yokomizo, K., Uyeda, M., 2000. Anti-herpes virus activity of Solanum steroidal glycosides. Biol. Pharma. Bull. 23, 363-364.
Jentsch, T.J., Stein, V., Weinreich, F., Zdebik, A.A., 2002. Molecular structure and physiological function of chloride channels. Physiol. Rev. 82, 503-568.
Lee, H.J., Song, LG, Yun, H.J., Jo, D.Y., Kim, S., 2014. CXC chemokines and chemokine receptors in gastric cancer: from basic findings towards therapeutic targeting. World J. Gastroenterol. 20, 1681-1693.
Lu, B., Yin, L, Xu, L, Peng, J., 2011. Application of proteomic and bioinformatic techniques for studying the hepatoprotective effect of dioscin against C[Cl.sub.4]-induced liver damage in mice. Planta Med. 77, 407-415.
Lv, L., Zheng, L, Dong, D., Xu, L., Yin, L, Xu, Y., Qi, Y., Han, X., Peng, J., 2013. Dioscin, a natural steroid saponin, induces apoptosis and DNA damage through reactive oxygen species: a potential new drug for treatment of glioblastoma multiforme. Food Chem. Toxicol. 59, 657-669.
Mondal, J., Panigrahi, A.K., Khuda-Bukhsh, A.R., 2014. Anticancer potential of Conium maculatum extract against cancer cells in vitro: Drug-DNA interaction and its ability to induce apoptosis through ROS generation. Pharmacogn. Mag. 10, S524-S533.
Schildberg, C.W., Croner, R., Schellerer, V., Haupt, W., Schildberg, F.W., Schildberg, M., Hohenberger, W., Horbach, T., 2012. Differences in the treatment of young gastric cancer patients: patients under 50 years have better 5-year survival than older patients. Adv. Med. Sci. 57, 259-265.
Wang, C.L, Liu, C., Niu, L.L., Wang, L.R., Hou, L.H., Cao, X.H., 2013a. Surfactin-induced apoptosis through ROS-ERS-Ca2*-ERK pathways in HepG2 cells. Cell Biochem. Biophys. 67, 1433-1439.
Wang, L., Meng, Q., Wang, C., Liu, Q., Peng, J., Huo, X., Sun, H., Ma, X., Liu, K., 2013b. Dioscin restores the activity of the anticancer agent adriamycin in multidrug-resistant human leukemia K562/adriamycin cells by down-regulating MDR1 via a mechanism involving NF-kappaB signaling inhibition. J. Nat. Prod. 76, 909914.
Wang, M.Y., Chen, P.S., Prakash, E., Hsu, H.C., Huang, H.Y., Lin, M.T., Chang, K.J., Kuo, M.L., 2009. Connective tissue growth factor confers drug resistance in breast cancer through concomitant up-regulation of Bcl-xL and cIAPl. Cancer Res. 69, 3482-3491.
Wang, Z., Cheng, Y., Wang, N., Wang, D.M., Li, Y.W., Han, F., Shen, J.G., Yang de, P., Guan, X.Y., Chen, J.P., 2012. Dioscin induces cancer cell apoptosis through elevated oxidative stress mediated by downregulation of peroxiredoxins. Cancer Biol. Ther. 13,138-147.
Weist, S., Eravci, M., Broedel, 0., Fuxius, S., Eravci, S., Baumgartner, A., 2008. Results and reliability of protein quantification for two-dimensional gel electrophoresis strongly depend on the type of protein sample and the method employed. Proteomics 8, 3389-3396.
Xiao, W., Jiang, Y., Men, Q., Yuan, L, Huang, Z., Liu, T., Li, W., Liu, X., 2015. Tetrandrine induces G1/S cell cycle arrest through the ROS/Akt pathway in EOMA cells and inhibits angiogenesis in vivo. Int. J. Oncol. 46, 360-368.
Xu, D., Li, Y., Li, X., Wei, L.L., Pan, Z., Jiang, T.T., Chen, Z.L., Wang, C., Cao, W.M., Zhang, X., Ping, Z.P., Liu, C.M., Liu, J.Y., Li, Z.J., Li, J.C., 2015. Serum protein S100A9, SOD3, and MMP9 as new diagnostic biomarkers for pulmonary tuberculosis by iTRAQ- coupled two-dimensional LC-MS/MS. Proteomics 15, 58-67.
Xu, L.N., Wei, Y.L., Dong, D.S., Yin, L.H., Qi, Y., Han, X., Xu, Y.W., Zhao, Y.Y., Liu, KX, Peng, J.Y., 2014. iTRAQ-based proteomics for studying the effects of dioscin against nonalcoholic fatty liver disease in rats. RSC Adv. 4, 30704.
Zhang, H., Li, H., Yang, L, Deng, Z., Luo, H., Ye, D., Bai, Z., Zhu, L, Ye, W., Wang, L., Chen, L., 2013. The C1C-3 chloride channel associated with microtubules is a target of paclitaxel in its induced-apoptosis. Sci. Rep. 3, 2615.
Xinwei Zhao (a),(1), Lina Xu (a),(1), Lingli Zheng (b), Lianhong Yin (a), Yan Qi (a), Xu Han (a), Youwei Xu (a), Jinyong Peng (a),*
(a) College of Pharmacy, Dalian Medical University, Western 9 Lvshunnan Road, Dalian 116044, China
(b) Department of Pharmaceuticals, The First Affiliated Hospital of Dalian Medical University, Dalian 116011, China
Abbreviations: iTRAQ, isobaric tags for relative and absolute quantitation; MTT, 3-(4, 5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide; AO, acridine orange; EB, ethidium bromide; DAPI, 4,6-diamidino-2-phenylindole; SCGE, single cell gel electrophoresis assay; TEM, transmission electron microscopy; ROS. reactive oxygen species; siRNA, small interfering RNA; Bd-2, B-cell leukemia 2 protein; Bax, Bcl-2- associated X protein; caspase-3, cysteinyl aspartate specific protease-3; caspase-9, cysteinyl aspartate specific proteinase-9; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; p-ERK, type I transmembrane ER-resident protein kinase; JNK, Jun N-terminal kinase; p-JNK, phospho-cjun specific protein kinase. RBM-3, RNA-binding motif protein 3; CLiC-3, chloride intracellular channels protein 3; GALR-2, Galanin receptor protein 2; CAP-1, adenylate cyclase-associated protein 1.
* Corresponding author. Tel.: +86 411 8611 0411; fax: +86 411 8611 0411.
E-mail address: firstname.lastname@example.org (J. Peng).
(1) These authors contributed same work to this paper and they are the co-first authors.
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|Author:||Zhao, Xinwei; Xu, Lina; Zheng, Lingli; Yin, Lianhong; Qi, Yan; Han, Xu; Xu, Youwei; Peng, Jinyong|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Mar 15, 2016|
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