Identification of an antitumor effect of demethylzeylasteral on human gastric cancer cells
- Authors:
- Published online on: November 18, 2020 https://doi.org/10.3892/ol.2020.12310
- Article Number: 49
Abstract
Introduction
The global burden of cancer continues to increase due to a variety of factors (1). Gastric cancer is the fourth most common cancer worldwide and is the second most common cause of cancer-associated mortality (2). The treatment of gastric cancer includes surgery, chemotherapy, radiotherapy and molecular targeted therapy, among which surgery combined with chemoradiotherapy is the most effective treatment regimen (3). However, the treatment has entered a bottleneck period due to congenital or acquired drug resistance and postoperative recurrence (4). Therefore, identification of new targets and signaling pathways related to the progression of gastric cancer may be beneficial for the treatment of gastric cancer.
Tumor occurrence and development are closely associated with uncontrolled cell proliferation, while malignant cells often escape apoptosis to obtain unlimited proliferation capacity (5). In this case, three types of evasion mechanisms of apoptosis exist, including the weakening of caspase function, the damage of the death receptor pathway, and the destruction of the balance between anti-apoptotic and pro-apoptotic proteins (6). Therefore, targeting cancer cell apoptosis by modulating key proteins or enzymes in the apoptosis-related signaling pathway has become an area of focus in cancer research (7). Notably, a number of natural products have been shown to act in apoptosis signaling pathways involved in cancer cell death. Camptothecin, a quinoline alkaloid, induced apoptosis of human myeloid leukemia cells by upregulating proapoptotic proteins and downregulating cyclins (8). Matrine, an alkaloid derived from the Sophora flavescensait, promoted apoptosis of gastric cancer cells by boosting the pro-apoptotic proteins, altering the ratio of Fas/FasL and activating caspase-3 (9,10). These findings have led researchers to focus on developing new potential anticancer agents.
Demethylzeylasteral is a triterpene monomer extracted from tripterygium wilfordii, which has been widely used in the study of anti-inflammatory immune regulation, antifertility and estrogen metabolism regulation (11–14). In recent years, the anticancer properties of demethylzeylasteral have been continuously studied. Li et al (15) reported that demethylzeylasteral significantly impeded the invasion of triple-negative breast cancer by blocking the TGF-β signaling pathway (15). Meanwhile, this compound may suppress glioma growth by mediating the miR-30e-5p/MYBL2 axis (16). Notably, demethylzeylasteral was found to evoke the apoptosis of melanoma cells by downregulating the level of MCL1 (17). However, the anticancer activity of demethylzeylasteral on gastric cancer cells and its underlying mechanism have not been investigated.
The present study aimed to determine and characterize the anticancer properties of demethylzeylasteral on human gastric cancer cells. The present study demonstrated that demethylzeylasteral impeded the viability and migration of gastric cancer cells, while inducing cancer cell apoptosis. Furthermore, treatment with demethylzeylasteral attenuated the ERK1/2 pathway, as well as a decreasing the levels of phosphorylated Akt (p-Akt) and phosphorylated GSK3β (p-GSK3β) in the cancer cells. The present study demonstrated that demethylzeylasteral has a therapeutic potential for gastric cancer.
Materials and methods
Reagents
The reagents used in this study were purchased as follows: Demethylzeylasteral (CAS:107316-88-1) from Target Molecule Corp.; fetal bovine serum (FBS) from Shanghai Nuova Pharmaceutical Technology Co., Ltd.; 0.25% Trypsin-EDTA, RPMI-1640 medium and penicillin/streptomycin from Gibco; Thermo Fisher Scientific, Inc.; dimethyl sulfoxide (DMSO) and enhanced chemiluminescent (ECL) substrate from Thermo Fisher Scientific, Inc.; Wright-Giemsa Stain it from Jian Cheng Technology Company; crystal violet from Sangon Biotechnology; Annexin V-FITC/PI apoptosis detection kit from Multi Sciences (Lianke) Biotech, Co., Ltd.; phosphate-buffered solution (PBS), mitochondrial membrane potential (MMP) assay kit with JC-1, and Cell Counting Kit-8 (CCK-8) from Beyotime Institute of Biotechnology; methanol and ethanol from Macklin Reagent Co., Ltd.; primary antibodies against Bax, cleaved PARP, caspase-9, cleaved caspase-3, Bax, c-Myc, GSK-3β, p-GSK-3β (Ser9), ERK1/2, phosphorylated ERK1/2 (p-ERK1/2), Akt, p-Akt and β-actin from Cell Signaling Technology, Inc.; horseradish peroxidase-conjugated secondary antibodies against rabbit and mice from Jackson ImmunoResearch Inc.
Demethylzeylasteral liquid preparation
A total of 10 mg demethylzeylasteral was dissolved in DMSO for preparation of 100 mmol/ml solution, and the solution was kept at −20°C for a long-term storage. For the experiments, the solution was diluted with DMSO to the indicated concentrations.
Cell culture
Human gastric cancer MKN-45 cells were purchased from American Type Culture Collection. Human normal gastric mucosal GES-1 cells were obtained from the Cell Bank of the Chinese Academy of Sciences. The cells were grown in a 37°C incubator with 5% CO2 and cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin for 24 h.
Cytotoxicity assay
CCK-8 was used to assess the cytotoxicity of the compound. In brief, MKN-45 cells were seeded onto 96-well plates (5,000 cells/well) and incubated with 0.2 µl DMSO or demethylzeylasteral at various concentrations (50 nM, 100 nM, 500 nM, 1 µM, 5 µM, 10 µM, 30 µM, 50 µM or 100 µM) for 24 h. Next, 10 µl CCK-8 solution was applied to each well, followed by incubation for 2 h at 37°C. Finally, the absorbance at a wavelength of 450 nm was measured using a microplate reader. Similarly, the effects of different concentrations of demethylzeylasteral on the activity of human normal gastric mucosa GES-1 cells were also investigated using the same method.
Colony formation assay
A colony formation assay was utilized to assess cell proliferation. MKN-45 cells in a single cell suspension state were seeded onto a 6-well plate at a density of 500 cells per well, and then treated with 2 µl DMSO or demethylzeylasteral at different concentrations (100 nM, 500 nM, 1 µM or 10 µM). The cells were grown in RPMI-1640 medium containing 10% FBS for 1 week until each colony reached 100–200 cells. Colonies were fixed in 100% methanol at room temperature for 20 min and stained using Giemsa staining at room temperature for 20 min. Finally, the plate was washed moderately with running tap water, and the colony with at least 100 cells was counted. Digital photography of colonies was conducted, and the data was analyzed using Prism software 7.0 (GraphPad Software, Inc.).
Transwell assay
The Transwell assay was used to assess the effect of demethylzeylasteral on gastric cancer cell migration. A total of 200 µl serum-free cell suspension containing 2×105 MKN-45 cells and 600 µl RPMI-1640 medium with 5% FBS were added to the upper chamber of the transwell chambers (8-µm pore size, Corning Inc.) and lower compartment of the Transwell chamber, respectively. Next, 2 µl DMSO or demethylzeylasteral at different concentrations (1, 3 or 10 µM) were applied to the upper chamber. After growth for 24 h, the cells migrating to the submembrane surface were fixed with methanol for 20 min at room temperature and stained with 0.5% crystal violet for 20 min at room temperature. Finally, the stained cells were photographed under a Leica optical microscope at ×100 magnification and ImageJ computer software 1.6 (National Institutes of Health) was used to calculate the area of cell migration.
Measurement of MMP
Alteration in MMP in apoptosis was investigated using an MMP-specific fluorescent probe, 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethyl-benzimidazole-carbocyanide iodine (JC-1; Beyotime Institute of Biotechnology). In brief, MKN-45 cells were treated with 2 µl DMSO or demethylzeylasteral at different concentrations (1, 3 or 10 µM) for 24 h and centrifuged for 5 min at −4°C and 500 × g. The cells were stained with JC-1 staining solution for 20 min in the dark at 37°C. After being washed twice with JC-1 buffer, the MKN-45 cells were re-suspended in JC-1 buffer and analyzed on a flow cytometer (BD FACSVerse™).
Apoptosis assay
Annexin V-FITC/PI apoptosis detection kit was used to assay apoptosis in MKN-45 cells treated with demethylzeylasteral, according to the manufacturer's protocol. MKN-45 cells were seeded onto 12-well plates (5×105 cells/well) and grown at 37°C overnight. Next, the cells were incubated with 2 µl DMSO or demethylzeylasteral at different concentrations (1, 3 or 10 µM) for 24 h. Following treatment, the cells were harvested by centrifugation (5 min at −4°C and 500 × g), rinsed twice with PBS and re-suspended in 500 µl Annexin-binding buffer. Finally, the cells were stained with 5 µl Annexin-V-FITC and 10 µl PI for 15 min at 4°C, and the fluorescence was determined using a flow cytometer (BD FACSVerse™; BD Biosciences). FlowJo 7.6 computer software (BD Biosciences) was used to analyze the data.
Western blot analysis
Protein expression was detected using western blot analysis. MKN-45 cells were seeded onto 6-well plates (1×106 cells/well) and treated with 2 µl DMSO or demethylzeylasteral at different concentrations for 24 h at 37°C. Following treatment, MKN-45 cells were washed twice with cold PBS, mixed with the loading buffer in each well, and then heated at 99°C for 10 min. Protein lysates were separated by electrophoresis on 12 or 15% SDS-PAGE, and then transferred onto nitrocellulose membranes. After being blocked with fresh 5% skimmed milk at room temperature for 2 h, the membranes were incubated with primary antibodies (Cell Signaling Technology, Inc.; 1:1,000) against Bax (cat. no. 5023), cleaved PARP (cat. no. 5625), cleaved caspase-9 (cat. no. 52873), cleaved caspase-3 (cat. no. 9669), Bcl-2 (cat. no. 15071), c-Myc (cat. no. 13987), GSK-3β (cat. no. 9315), p-GSK-3β (Ser9; cat. no. 5558), ERK1/2 (cat. no. 4695), p-ERK1/2 (cat. no. 4376), Akt (cat. no. 4685), p-Akt (cat. no. 9563) and β-actin (cat. no. 8457) at 4°C overnight. The membranes were subsequently incubated with horseradish peroxidase-conjugated secondary antibodies against rabbit and mice at room temperature for 2 h. The target proteins in the membrane were detected and visualized using the Chemiluminescence Luminol kit. ImageJ software (National Institutes of Health) was used to measure the intensity of the bands.
Statistical analysis
Demethylzeylasteral structure was drawn using ChemDraw Professional 16.0 software (PerkinElmer, Inc.). All statistical data are expressed as the mean ± standard error of the mean, and all experiments were repeated at least three times. GraphPad Prism 7.0 was used to perform Student's t-tests or one-way analysis of variance with a Dunnett's post hoc test to analyze the significance of the results. P<0.05 was considered to indicate a statistically significant difference.
Results
Demethylzeylasteral has cytotoxic effects on human gastric cancer cells
The chemical structure of demethylzeylasteral was depicted in Fig. 1A. CCK-8 assay was applied to test whether demethylzeylasteral has a cytotoxic effect on human gastric cancer MKN-45 cells. As shown in Fig. 1B, demethylzeylasteral exhibited a concentration-dependent cytotoxic effect on MKN-45 cells. Additionally, the 50% inhibitory concentration (IC50) values were found to be 8.174 µM for the MKN-45 cells. In the experiments, human gastric mucosa GES-1 cells were included as a control. As shown in Fig. 1C, the CCK-8 assay revealed that demethylzeylasteral at concentrations below 10 µM (0.1, 0.5, 1 or 5 µM) had no significant influence on the survival rate of GES-1 cells, while cytotoxicity was evident in the control cells treated with 10 µM or higher concentrations of demethylzeylasteral (10, 30 or 50 µM).
Demethylzeylasteral inhibits gastric cancer cell proliferation
In the present study, MKN-45 cells were treated with various concentrations of demethylzeylasteral for 1 week. As shown in Fig. 2A, the treatment decreased colony formation in all tested gastric cancer cells. Furthermore, quantitative analysis of the colonies indicated that demethylzeylasteral decreased the proliferation of gastric cancer cells in a dose-dependent manner.
Demethylzeylasteral suppresses cancer cell migration
The effects of demethylzeylasteral on the migration of human gastric cancer cells were investigated using an in vitro Transwell assay. As shown in Fig. 2C, a significant decrease in the number of cells migrating to the lower surface of the filter was observed in the group of MKN-45 cells treated with demethylzeylasteral, compared with that in the DMSO-treated group. Further statistical analysis revealed that treatment with demethylzeylasteral at various concentrations suppressed the migration of MKN-45 cells in a dose-dependent manner (Fig. 2D).
Demethylzeylasteral induces apoptosis of MKN-45 cells
It has been reported that numerous compounds inhibit tumor cell growth by inducing apoptosis (18). To determine whether the anti-proliferative effect of demethylzeylasteral is associated with apoptosis, Annexin V-FITC/PI double staining and flow cytometry were used to detect the number of apoptotic cells. As shown in Fig. 3A and B, compared with the control group, treatment with demethylzeylasteral at various concentrations for 24 h resulted in a significant increase in the rate of apoptosis from 11.15% (DMSO) to 28.6% (10 µM). Notably, the number of apoptotic cells was gradually increased along with increasing concentrations of demethylzeylasteral (Fig. 3B). Furthermore, western blot analysis revealed that markedly upregulated expression of pro-apoptotic proteins, including Bax, cleaved caspase-9, cleaved caspase-3 and cleaved PARP, as well as downregulated expression of an anti-apoptotic protein, Bcl-2, were detected in MKN-45 cells treated with demethylzeylasteral (Fig. 4). These observations indicated that demethylzeylasteral may induce apoptosis of MKN-45 cells.
Demethylzeylasteral affects the MMP in MKN-45 cells
It has been shown that the cleavage of caspase-3, and caspase-9 and PARP is increased during the mitochondria-dependent apoptosis (19). JC-1 staining was used to investigate whether demethylzeylasteral promotes apoptosis via the mitochondrial-dependent pathway. As shown in Fig. 5A and B, the JC-1 polymer was significantly decreased in MKN-45 cells treated with demethylzeylasteral at various indicated concentrations for 24 h. Furthermore, the change in JC-1 from red to green fluorescence indicated a loss of the MMP (Fig. 5A). Taken together, these results suggested that demethylzeylasteral-induced apoptosis in the cancer cells may involve the mitochondria-dependent pathway.
Demethylzeylasteral treatment downregulates Akt/GSK3β pathway
Akt is a canonical oncogenic kinase serving a prominent role in tumor progression (20). To improve understanding of the molecular mechanism underlying the antitumor effect of demethylzeylasteral on the cancer cells, the levels of total Akt, total GSK-3β, p-Akt and p-GSK-3β (ser9) in MKN-45 cells treated with demethylzeylasteral were investigated. As depicted in Fig. 6A, treatment with increasing concentrations of demethylzeylasteral led to a stepwise decrease in the levels of p-Akt and p-GSK-3β, while the levels of total Akt and GSK-3β remained unchanged. Quantitative analysis revealed a dose-dependent decrease in the levels of p-Akt and p-GSK-3β (Fig. 6B and C).
Demethylzeylasteral attenuates the ERK1/2 pathway
It has been documented that the activation of the MAPK pathway contributes toward the development of gastric cancer. To clarify whether the anticancer properties of demethylzeylasteral are associated with MAPK signaling pathways in gastric cancer cells, the expression of JNK, ERK1/2 and p38 protein in demethylzeylasteral-treated cells was measured using western blot analysis. The analysis revealed that, compared with the DMSO-treated cells, the level of p-ERK1/2 was decreased in the cancer cells treated with demethylzeylasteral, while no significant change in the levels of phosphorylated-JNK and phosphorylated-p38 was detected in the cells treated with various concentrations of demethylzeylasteral (Fig. 7). ERK1/2 serves a pro-tumorigenic role in numerous cancer types and has hundreds of substrates (21), some of which control cell growth, differentiation, survival and death by regulating the phosphorylation and activation of transcription factors. Given that c-Myc is a downstream target of ERK1/2 (22,23), the present study aimed to analyze the expression of c-Myc protein in MKN-45 cells incubated with demethylzeylasteral. As shown in Fig. 7A, treatment with demethylzeylasteral decreased the expression of c-Myc, while downregulating p-ERK1/2 in the cancer cells. Taken together, these data suggested that the ERK pathway may be involved in demethylzeylasteral-mediated cell proliferation and apoptosis in MKN-45 cells.
Discussion
Gastric cancer is the second leading cause of cancer-associated mortality after lung cancer. Although the rate of early diagnosis has been improved, the therapeutic outcomes of gastric cancer requires improvement due to postoperative recurrence, acquired drug resistance and late metastasis (4,24). Therefore, developing novel drugs for the treatment of gastric cancer may contribute toward promoting the therapeutic outcomes. Demethylzeylasteral is a natural compound from tripterygium wilfordii, which possesses multiple pharmacological effects. Although this compound has been shown to be associated with breast cancer, glioma and malignant melanoma, determining whether it has a broad-spectrum antitumor effect requires further investigation. The present study aimed to investigate the potential anticancer effect of demethylzeylasteral on gastric cancer cells.
The present study investigated the anti-proliferative activity of demethylzeylasteral by using gastric cancer MKN-45 cell line. The CCK-8 assay demonstrated that demethylzeylasteral had a dose-dependent cytotoxic effect on cancer MKN-45 cells. Demethylzeylasteral was found to downregulate the Akt/GSK-3β pathway, as well as the ERK1/2 pathway. Furthermore, it was found that treatment with demethylzeylasteral decreased MMP, while downregulating Bcl-2 expression and upregulating Bax, cleaved caspase-9, cleaved caspase-3 and cleaved PARP expression.
Apoptosis or programmed cell death is mediated by the endogenous mitochondrial pathway and the exogenous death receptor pathway (25). The complex mechanism involves numerous signaling pathways (6); among them, the intrinsic pathway of mitochondria-induced apoptosis is dependent on the expression of a series of proteins, including the Bcl-2 family, which contains anti-apoptotic and pro-apoptotic proteins. Bcl-2 family proteins mediate the activation of cysteine aspartic acid specific proteases (caspases) for inducing apoptosis (26–28). Caspases are the core of apoptosis mechanism, as they act as both the promoters and the executors of cell death (29). Once activated, the initiator caspases will cleave and activate executioner caspases, which subsequently perform critical cleavage on specific cell substrates, ultimately leading to apoptosis (30). Activated Caspase-9 in the endogenous mitochondria-dependent pathway in turn results in the activation of Caspase-3 (31). Caspase-3 is a key component in the caspase-dependent apoptosis pathway, which triggers the cleavage of downstream substrate PARP (32), leading to chromatin lysis and apoptosis (18). In the present study, treatment of MKN-45 cells with demethylzeylasteral caused an upregulation in the levels of cleaved Caspase-9, cleaved Caspase-3 and cleaved PARP; a decreased expression of anti-apoptotic protein Bcl-2; increased expression of pro-apoptotic protein Bax; and disruption of MMP. All these data suggested that demethylzeylasteral-elicited apoptosis in MKN-45 cells may involve the activation of the caspase cascade in the endogenous death pathway (mitochondrial pathway).
Akt, a serine/threonine kinase, is highly amplified in gastric cancer and regulates numerous biological and pathological processes, including apoptosis, cell proliferation and glucose usage (33,34). Apatinib is known to be significant for the treatment of advanced gastric cancer (35). Compound Astragalus polysaccharide (AsPs) works with apatinib against gastric cancer by inhibiting the Akt pathway in AGS cells (36). In addition, helicobacter pylori (HP) has been highlighted due to its association with and involvement in the occurrence of gastric cancer associated with the Akt/GSK-3β signaling pathway (37). Additionally, clinical studies have demonstrated that Akt phosphorylation is involved in tumor invasion, and p-Akt status may be associated with early recurrence and poor prognosis (38). Akt promotes survival and cell cycle by phosphorylating cellular proteins, including GSK-3α and GSK-3β (39); GSK-3α and GSK-3β constitute two main isoforms of GSK-3, a multifunctional serine/threonine protein kinase. It has been reported that GSK-3 is regulated by activated Akt, and activated GSK-3 (non-phosphorylated state) regulates cell cycle and apoptosis (40). It is worth noting that GSK-3β is inactivated once phosphorylated on serine 9. Multiple studies have demonstrated that Dioscin, β-sitosterol and Isobavachalcone, respectively, induced apoptosis of osteosarcoma, pancreatic cancer and colon cancer cells by downregulating Akt/GSK-3β pathway (41–43). The present study demonstrated that the treatment of MKN-45 cells with demethylzeylasteral in induced cell growth inhibition and apoptosis, while suppressing the Akt/GSK-3β pathway. These findings suggested that the anti-proliferative and apoptosis-inducing effects of demethylzeylasteral on gastric cancer cells are associated with the Akt/GSK-3β signaling pathway.
The MAPK signaling pathway includes JNK, ERK and p38, and serves a considerable role in cell proliferation, survival and apoptosis (44). The present study demonstrated that significantly decreased p-ERK1/2, but minor changes in the phosphorylation of JNK or p38, were detected in the cancer cells treated with demethylzeylasteral. ERK 1/2 is a key modulator of cell proliferation, and inhibitors of the ERK pathway are currently used as potential anticancer agents in clinical trials (45). Furthermore, ERK1/2 phosphorylation-targeting compounds were found to serve a vital role in the survival of gastric cancer. Wang et al (46) reported that 1,4-naphthoquinone derivatives evoked apoptosis of gastric cancer cells by downregulating the ERK pathway (46). An extract from Triptolide has been identified to have antitumor effects on gastric cancer cells (47). Furthermore, recent studies have shown that Triptolide may prevent the proliferation and metastasis of esophageal cancer cells via the ERK1/2 pathway (48). The aforementioned two compounds differ from demethylzeylasteral in chemical properties and are greatly limited in clinical application due to their undesirable side effects. Once translocated to the nucleus, p-ERK1/2 activates multiple transcription factors, including CREB, NF-κB and c-Myc, thereby regulating various cellular processes, including cell survival and proliferation (22,23). The present study observed a dose-dependent inhibitory effect of demethylzeylasteral on the expression of p-ERK 1/2 in cancer cells. Meanwhile, treatment with demethylzeylasteral significantly decreased the expression level of c-Myc. We hypothesize that demethylzeylasteral inhibits gastric cancer cell proliferation and induces apoptosis of cancer cells by modulating the ERK1/2 pathway and downregulating c-Myc expression.
Toxicity has long been a key factor limiting the clinical application of novel research drugs. The growing advancement in technology suggests that the toxicity of demethylzeylasteral may be decreased through either increasing its solubility via modification of the chemical structure or administration combined with other chemotherapy drugs. More importantly, structural and chemical modification of demethylzeylasteral will enable more in vivo experiments to be conducted in future studies. Therefore, decreased toxicity may make it possible for demethylzeylasteral to become a novel drug for the treatment of gastric cancer.
In conclusion, the results of the present study suggested that demethylzeylasteral inhibited the proliferation and migration of gastric cancer cells, while promoting the apoptosis of cancer cells via the mitochondria-dependent pathway. Further investigation suggested that the Akt/GSK-3β and ERK1/2 signaling pathways may be involved in the demethylzeylasteral-induced antitumor effects on the cancer cells (Fig. 8). These results suggested that demethylzeylasteral has therapeutic potential for the treatment of gastric cancer.
Acknowledgements
Not applicable.
Funding
This work was supported by The Natural Science Research Key Project of Education Office of Anhui Province (grant no. KJ2019A0329) and Research and Innovation Team of Bengbu Medical College (grant no. BYKC201908).
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
Authors' contributions
YY, ZW and FQ designed the study. YY, MZ and TH collected experimental materials and carried out the experiments. FS performed the statistical analyses. YY wrote the manuscript. FQ and ZW made critical revisions to the manuscript. ZW given final approval of the version to be published. All authors read and approved the final version.
Ethics approval and consent to participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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