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Astragalus polysaccharide enhanced antitumor effects of Apatinib in gastric cancer AGS cells by inhibiting AKT signalling pathway
Biomedicine & Pharmacotherapy 100 (2018) 176–183
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Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Astragalus polysaccharide enhanced antitumor effects of Apatinib in gastric cancer AGS cells by inhibiting AKT signalling pathway
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Jun Wua, Junxian Yub, Jing Wanga, Chenguang Zhangc, Kun Shanga, Xiaojun Yaod, , ⁎ Bangwei Caoa, a
Cancer Center, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China Department of Pharmacy, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China c Department of Biochemistry and Molecular biology, Capital Medical University, Beijing 100069, China d State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau, 999078, China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Gastric cancer Apatinib AsPs AKT
Apatinib has been proved effective in the treatment of advanced gastric cancer. Traditional Chinese medicine is often considered as adjuvants which could increase the effects and counteract the side effects of chemotherapy. The present study aims to explore the antitumor effects of Astragalus polysaccharide (AsPs) in combination with Apatinib in gastric cancer AGS cells. Our results demonstrated that the expression of VEGFR-2 was observed in human gastric cancer line AGS. Both Apatinib and AsPs could significantly inhibit the proliferation of AGS cells in a dose-dependent manner and Apatinib in combination with AsPs showed enhanced inhibitory effects on cell proliferation, migration and invasion compared with Apatinib monotherapy. Moreover, there was a remarkable increase in apoptosis following Apatinib treatment which could be enhanced by the addition of AsPs. Western blotting showed that the combination of Apatinib and AsPs could inhibit the expression of phosphorylated AKT (p-AKT) and MMP-9 expression. In addition, both Apatinib alone and Apatinib in combination with AsPs induced celluar autophagy which could be attenuated by the autophagy inhibitor 3-MA. The suppression of autophagy leaded to further apoptosis induction and cell proliferation suppression. In conclusion, the current study showed AsPs enhanced antitumor effects of Apatinib on AGS cells by the mechanism which includes inhibition of AKT signaling pathway. Apatinib-induced autophagy could be attenuated by 3-MA, which subsequently increased the apoptosis rate. On the basis of our study, the combination of Apatinib and AsPs could be considered as a potential candidate in the gastric cancer treatment.
1. Introduction Gastric cancer is one of the most malignant tumors worldwide and approximately half of the patients with gastric cancer are in China [1]. Despite a worldwide decline in incidence over the past few decades, most of the patients are still diagnosed at the advanced stage of illness due to the lack of early clinical symptoms and sensitive markers. Although many efforts have been made in gastric cancer treatment, the prognosis is still very poor with a median overall survival time (OS) less than 1year [2]. In the recent years, molecular-targeted therapy has become the research hotspot in cancer treatment. A number of targeted agents have shown effectiveness in cancer patients. Angiogenesis plays very important role in tumor development and metastasis. Vascular endothelial growth factor (VEGF) which binds to the high-affinity
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receptors (VEGFR) is a key mediator of angiogenesis [3]. A few angiogenesis inhibitors have shown efficacy in human tumors, such as in lung, breast and colorectal cancers [4–6]. Targeting the VEGF/VEGFR pathway is a viable alternative for gastric cancer treatment due to the high levels of VEGF/VEGFR expression which is associated with poor prognosis [7]. A systematic review and meta-analysis showed angiogenesis inhibitor-containing regimens were superior to routine regimens without angiogenesis inhibitor in patients with advanced gastric cancer [8]. Apatinib, a small-molecule tyrosine kinase inhibitor (TKI), specifically targets the intracellular ATP-binding domain of VEGFR-2, and subsequently inhibits angiogenesis by blocking the downstream signal transduction [9]. Apatinib was demonstrated effective in improving OS and progression-free survival (PFS) for patients with chemotherapy-
Corresponding author at: Cancer Center, Beijing Friendship Hospital, Capital Medical University, 95 Yongan Road, Xicheng District, Beijing 100050, China. Corresponding author. E-mail addresses: [email protected] (X. Yao), [email protected] (B. Cao).
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https://doi.org/10.1016/j.biopha.2018.01.140 Received 21 October 2017; Received in revised form 2 January 2018; Accepted 28 January 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
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of 5 μl PI solution to each group. Percentage of cell in the early and late stage of apoptosis was determined by flow cytometry.
refractory advanced or metastatic gastric cancers in the clinical phase III trial [10]. Chinese traditional medicine was used to treat a wide range of human diseases including cancers. It usually serves as adjuvants to increase the effects and/or counteract the side effects of chemotherapy [11,12]. Astragalus polysaccharide (AsPs), the active component extracted from Astragali Radix which is the root of Astragalus membranaceus Bunge [13], was reported to have antitumor effects by improving immunity, inducing apoptosis and inhibiting tumor growth. Moreover it was also shown to reduce the toxicity of chemotherapeutics [14,15]. In the present study, we investigated the enhanced antitumor effects of AsPs combined with Apatinib in gastric cancer cell. The alterations of cellular autophagy affected by Apatinib and AsPs were also explored.
2.4. Migration assay Cell migration was assessed by wound healing assay. AGS cells were seeded into 12-well plates. To create a “scratch’’, cell monolayers in each well were scratched in a straight line with a 200 μl-pipette tip when the cell confluency reached about 90%. We removed the debris and smoothed the edge of the scratch by washing the culture with PBS twice and then replaced with 1 ml DMEM containing different reagents without FBS for 24 h. The concentration of Apatinib and AsPs was 20 μg/ml and 200 μg/ml, respectively. It is important to create scratches of approximately similar width in the variously treated cells. The scratch lines were inspected at 0 and 24 h. Image fields were captured by using an inverted phase contrast microscope equipped with a digital camera (Olympus, Tokyo, Japan). By comparing the images from time 0 h to 24 h, we were able to obtain/calculated the distance of each scratch closure on the basis of the following formula: Cell motility = (distance 24 h - distance 0 h)/distance 0 h. The images acquired for each sample were further analyzed quantitatively by ImageJ Plus. Each experiment was performed in triplicate.
2. Materials and methods 2.1. Cell culture and reagents HUVEC (human umbilical vein endothelial cell) and human gastric cancer cell line AGS were acquired from the department of experimental research center in Beijing Friendship Hospital (Beijing, China). AGS and HUVEC were cultured in Dulbecco Modified Eagle Medium (DMEM, Corning, Virginia, VA, USA) supplemented with 10% FBS (fetal bovine serum, Biological Industries, Israel) and 1% penicillin/ streptomycin (KeyGen, Nanjing, China) in a 5% CO2 atmosphere at 37 °C. Apatinib was a gift of Jiangsu Hengrui medicine Co, Ltd (Jiangsu, China). AsPs was purchased from Nanjing Zelang Biotech Ltd (Nanjing, China). Both Apatinib and AsPs were dissolved by Dimethyl sulfoxide (DMSO), and both of them were prepared freshly prior to each test. 3Methyladenine (3-MA) was purchased from Sigma-Aldrich (St. Louis, MO, USA).
2.5. Invasion assay Transwell assay was performed to determine cell invasion. The coculture chambers (24-wells, 8-μm pore size, Costar, Corning Inc., ME, USA) were used in this section. In brief, prechilled upper inserts were coated with 60 μl Matrigel (Corning, USA) per well (1:20 in dilution) and then placed at 37 °C. The dilution agent was prechilled DMEM without FBS. After 6 h, upper inserts were hydrated by DMEM without FBS for 1 h at room temperature prior to addition of AGS cells. 3 × 104 cells per well suspended in 200 μL serum-free DMEM medium were added into the upper chamber and 600 μL DMEM medium containing 10% FBS was placed in the lower chamber. The concentration of Apatinib and AsPs was 20 μg/ml and 200 μg/ml. After 24 h incubation at 37 °C in a 5% CO2 atmosphere, the invaded cells left at the bottom of chambers were fixed in 4% paraformaldehyde for 25 min and then stained with 0.5% crystal violet (Macklin, Shanghai, China) for 15 min at room temperature. The cells at the upper layer were carefully removed with a cotton swab while the invaded cells at the lower layer were counted after air-drying. The crystal violet-positive cells were counted in 6 random visual fields for each chamber using an inverted phase contrast microscope (Olympus, Japan).
2.2. Cell proliferation assay MTS (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxypheny)-2(4-sulfophenyl) -2H- tetrazolium, innersalt), a CellTiter-96 AqueousOne Solution Cell Proliferation Assay Kit (Promega, Madison, WI, USA) was performed to detect cell proliferation. The cells were cultured in 96-well plates at a density of 8 × 103 cells per well, and then treated with DMEM containing reagents in different concentrations according to the experimental design when cells adhered overnight. Cells were treated with Apatinib in concentrations ranging from 0 to 30 μg/ml and AsPs from 0 to 600 μg/ml for 24 h. MTS solution (10 μl/well) was added to each experimental well and cultured for 1 h in a 5% CO2 atmosphere at 37 °C. The absorbance was measured on The ELx808™ Absorbance Microplate Reader (BioTek, Winooski, VT, USA) at the wave length of 490 nm. The viability of cell proliferation is directly proportional to A490 value. The concentration to inhibit cell proliferation by 50% (IC50) was calculated by using a linear regression equation. Triplicate experiments were performed in parallel manner for each concentration point.
2.6. Western blotting AGS cells were treated with reagents at indicated concentrations after 24 h incubation in 6-cm2 plates. The whole proteins were lysed in RIPA buffer (Applygen, Beijing, China) supplemented with protease inhibitor cocktail (Amresco, Ohio, USA) and the concentrations were quantified with a bicinchoninic acid (BCA) protein assay kit (Merck, Darmstadt, Germany). Protein samples were mixed with 5 × loading buffer (Applygen, Beijing, China) and heated at 95 °C for 10 min. An equal amount of 30 μg protein was separated by SDS-PAGE gel (sodium dodecyl sulfate- polyacrylamide gel electrophoresis) and then transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked with blocking buffer TBST (Tris-buffered saline and 0.2% Tween) containing 1.5% BSA (Bovine Serum Albumin) for 2 h at room temperature, and then incubated overnight with specific primary antibodies at 4 °C. Then the membranes were washed three times by TBST and incubated with HRP (horseradish peroxidase)-conjugated secondary antibody for 1 h at room temperature. LC3 rabbit anti-human monoclonal antibody (1:1000) and AKT rabbit anti-human monoclonal antibody (1:1000) were purchased from Cell Signaling Technology (Beverly, MA, USA). VEGFR-2 (1:1000) rabbit anti-human
2.3. Apoptosis assay Annexin V-FITC and propidium iodide (PI) staining assay were used to determine cell apoptosis by an apoptosis detecting kit (KeyGen, Nanjing, China) according to the manufacturer's instructions. AGS cells were plated and grown in 6-well plates in cultured standard growth conditions until they reached 70% confluency and then exposed to various treatments for 24 h. The concentration of Apatinib and AsPs was 20 μg/ml and 200 μg/ml, respectively. After being harvested by trypsin solution without EDTA (Ethylene Diamine Tetraacetic Acid), cells were washed with pre-chilled PBS (phosphate buffer saline) twice and then centrifuged at 2000 rpm/min for 5 min. The cell pellets were resuspended in binding buffer of 300 μl and incubated with 5 μl Annexin V-FITC on ice in the darkness for 15 min followed by addition 177
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3.4. AsPs enhanced the inhibition of cell migration and invasion caused by Apatinib
monoclonal antibody was purchased from Abcam (Cambridge, UK). MMP-9 (1:100) mouse anti-human monoclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); CST, USA). PAKT rabbit anti-human polyclonal antibody (1:500) was purchased from Abclonal (Wuhan, China). β-Actin rabbit anti-human monoclonal antibody (1:3000) as the interval control was obtained from Abcam (Cambridge, UK). The goat anti-rabbit and anti-mouse IgG conjugated to HRP antibody (1:5000; Santa Cruz Biotechnology Inc.) were used as the secondary antibody. Detection for visualization was performed by an enhanced chemiluminescence detection system (ECL, Millipore, USA).
Wound-healing and Transwell assay were conducted to determine the effects of Apatinib and AsPs on cell migration and invasion, respectively. Wound-healing assay results showed the Apatinib-treated group effectively inhibited cell migration (P < .05, Fig. 3A-B), while the AsPs-treated group did not affect the scratch closure significantly compared with the control group (P > .05). We also found Apatinib combined with AsPs inhibited cell migration to greater extent in comparison with Apatinib-treated group as shown in Fig. 3A-B (P < .05). Meanwhile, consistent with the wound-healing results, Transwell assay showed Apatinib decreased the invasive cell numbers significantly compared with the control group after statistical analysis as shown in Fig. 3C-D (P < .01). When Apatinib was combined with AsPs, inhibition in cell invasion to greater extent was observed compared with the Apatinib-treated group (P < .05). To explore the mechanisms behind the results above, MMP-9 protein expression was detected by western blotting. Results show MMP-9 expression was inhibited significantly by Apatinib after 24 h treatment as shown in Fig. 3E (P < .05), and the Apatinib + AsPs-treated group inhibited the expression of MMP-9 to greater extent. However, AsPs alone did not significantly inhibit MMP-9 expression as compared with the control group (P > .05, Fig. 3E).
2.7. Statistical analysis The data presented in this study were expressed as mean ± D. Statistical analysis and graphs were performed by GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA). Differences between groups were analysed using one-way analysis of variance (ANOVA). Values of P < .05 were considered statistically significant. 3. Results 3.1. VEGFR2 expression in AGS cell line
3.5. AsPs enhanced the inhibition of p-AKT expression
Firstly, AGS cell line was evaluated for VEGFR-2 expression by western blotting and HUVEC was used as the positive control for VEGFR-2 expression. Cells were cultured in 6-cm2 plates and the procedures of western blotting were described in the Materials and methods section. As shown in Fig. 1A, VEGFR-2 expression was detected in AGS cell line.
Western blotting was carried out to determine the effects of Apatinib and AsPs on p-AKT, which is the downstream effector molecule in the VEGFR-2 signaling pathway. After 24 h incubation, we found Apatinib decreased the level of p-AKT expression, and Apatinib combined with AsPs inhibited p-AKT expression in even greater extent (P < .05, Fig. 4A). However, compared with the control group, AsPs used at 200 μg/ml alone did not change p-AKT expression significantly (P > .05).
3.2. Apatinib and AsPs inhibited cell proliferation MTS results showed that with the increase of reagent concentration, either Apatinib or AsPs reduced A490 value as shown in Fig. 1B-C. As compared with the control group, Apatinib at 20 μg/ml and AsPs at 500 μg/ml displayed significant inhibiting effects on proliferation in AGS (P < .001). The IC50 values of Apatinib and AsPs were 26.3 μg/ml and 660.2 μg/ml, respectively. To clarify whether AsPs could increase the inhibitory effects of Apatinib on cell proliferation, we chose Apatinib at 20 μg/ml combined with AsPs at 50, 100, 200 and 400 μg/ml. Results revealed that Apatinib in combination with AsPs at 200 μg/ml produced stronger inhibition on cell proliferation compared with Apatinib used alone as shown in Fig. 1D (P < .01). Moreover, Apatinib at 20 μg/ml in combination with AsPs at 200 μg/ml decreased the IC50 values to 239.9 μg/ml. However, AsPs alone at 200 μg/ml did not display inhibitory effects (P > .05, Fig. 1C). Therefore, we chose the combination of Apatinib at 20 μg/ml and AsPs at 200 μg/ml to explore the mechanisms of AsPs exerting the enhanced antitumor effects in the subsequent experiment.
3.6. Apatinib and AsPs induced cellular autophagy To explore whether Apatinib and AsPs could change cellular autophagy level, LC3, a specific marker of autophagy was detected by western blotting. Cells were incubated with various concentration of Apatinib (0, 10, 20, 30 μg/ml) and AsPs (0, 200, 400, 600 μg/ml) for 24 h. Results showed either Apatinib or AsPs increased LC3 expression in a dose-dependent manner (P < .05, Fig. 4B,C). Apatinib at 20 μg/ml combined with AsPs at 200 μg/ml significantly increased LC3 expression compared with the Apatinib-treated group (Fig. 4D, P < .05). Therefore, the results indicate that both Apatinib and AsPs induced cellular autophagy. 3.7. Autophagy inhibitor 3-MA inhibited cell proliferation and increased apoptosis more significantly To determine the potential role of the increased autophagy induced by Apatinib and AsPs on cell proliferation, cells were pre-treated with autophagy inhibitor 3-MA (300 μg/ml) for 1 h, followed by various treatments for 24 h. The concentration of Apatinib and AsPs was 20 μg/ ml and 200 μg/ml, respectively. MTS assay showed Apatinib and Apatinib + AsPs in combination with 3-MA significantly inhibited cell proliferation as compared to the single use of above compounds (Fig. 5A, P < 0.05). Furthermore, AsPs + 3-MA and 3-MA monotherapy showed no obvious effect on cell proliferation (Fig. 5A, P > .05). To clarify the effects of 3-MA on cell apoptosis, Annexin VFITC and PI staining assay were used to detect cell apoptosis, and the percentage of apoptosis was also obtained by flow cytometry. As shown in Fig. 5B-C, we found that Apatinib + 3-MA (17.7 ± 3.2%) and Apatinib + AsPs + 3-MA (23.2 ± 3.7%) significantly increased cell apoptosis compared with them used singly (Apatinib: 13.3 ± 2.9%;
3.3. AsPs combined with Apatinib increased cell apoptosis To reveal the effects of Apatinib and AsPs on cell apoptosis, Annexin V and PI staining assay were performed to detect apoptosis rate of AGS cells treated with various reagents for 24 h and then the apoptosis percentage was analyzed by flow cytometry. As shown in Fig. 2, the percentage of apoptotic cells increased from 6.9 ± 3.1% in the control group to 12.5 ± 2.9% in the Apatinib-treated group (P < .01). We observed the Apatinib + AsPs-treated group significantly increased apoptosis percentage compared with the Apatinib-treated group (19.7 ± 3.4%:12.5 ± 2.9%, P < .01). However, compared with the control group, AsPs at 200 μg/ml in a single use did not enhance apoptosis percentage (5.8 ± 2.6%:6.9 ± 3.1%, P > .05). Therefore, we concluded that Apatinib induced cell apoptosis and AsPs enhanced apoptosis induced by Apatinib. 178
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Fig. 1. Expression of VEGFR-2 in AGS. HUVEC was used as the positive control for VEGFR-2, Western blotting showed VEGFR-2 was detected in AGS (A). AsPs enhanced the inhibitory effects of Apatinib on cell proliferation. MTS showed either Apatinib or AsPs reduced A490 value in a dose-dependent manner after 24 h treatment (B-C). Apatinib at 20 μg/ml in combination with AsPs at 200 μg/ml showed stronger inhibition on cell proliferation compared with the single use of Apatinib group (D). *P < .05, **P < .01, ***P < .001 vs control; ## P < .01, ###P < .001 vs Apatinib 20 μg/ml.
Apatinib + AsPs: 16.8 ± 3.3%; P < .05), (8.9 ± 2.6%) did not increase cell apoptosis to pared with AsPs alone (8.1 ± 2.5%; P > .05). control group (5.6 ± 2.3%), 3-MA alone (7.1 ± cell apoptosis (P > .05).
Apatinib, a novel oral antiangiogenic agent, was approved as a third-line treatment for gastric cancer patients. In our study, Apatinib was proved effective in inhibiting cell proliferation, migration and invasion of AGS cell line. In addition it also induced cell apoptosis. Our findings are consistent with the results of a new study that showed Apatinib inhibited cell viability, migration and invasion capabilities and increased apoptosis percentage in colon cancer cells [17]. However, drug resistance remains a major problem in tumor therapy including antiangiogenic therapies [18]. Long-term VEGF/VEGFR2 inhibition like bevacizumab (VEGF monoclonal antibody) was found to lead to the upregulation of other tumor-derived angiogenic factors, including platelet-derived growth factor (PDGF), angiopoietin (Ang), hepatocyte growth factor (HGF), and epidermal growth factor (EGF), which contributed to the mechanism of acquired resistance [19]. So, we infer Apatinib probably face the same difficulty in the years to come. At present, due to its little toxicity, Chinese traditional medicine is usually
but AsPs + 3-MA greater extent comCompared with the 2.1%) did not affect
4. Discussion According to the latest cancer statistics in China, gastric cancer is identified as the second leading cause of cancer incidence and mortality, posing a huge threat to public health [16]. Conventional chemotherapy or postoperative adjuvant chemotherapy is beneficial to gastric cancer patients, but some patients could not bear it because of its multiple adverse effects, resulting in discontinuation of treatment. In recent years, a series of antiangiogenic agents have been developed and improved prognosis of gastric cancer patients in clinical trials [8]. 179
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Fig. 2. AsPs increased apoptosis induced by Apatinib in AGS. AGS exposed to control, Apatinib 20 μg/ml, AsPs 200 μg/ml and Apatinib 20 μg/ml + AsPs 200 μg/ml after 24 h followed by Annexin V-FITC and propidium iodide staining and apoptosis percentage was detected by flow cytometry. **P < .01, ***P < .001 vs control; ##P < .01 vs Apatinib.
AKT signaling pathway could prevent cell apoptosis and enhance cell proliferation and migration in human cancers [25]. We found that the p-AKT expression was inhibited by Apatinib. This could be additionally suppressed by the administration of AsPs. Based on our findings, we believe that AsPs potentiates the antitumor effects of Apatinib on AGS cells by blocking AKT signaling pathway. Autophagy is the fundamental process through which cells utilize lysosomal hydrolases to degrade cytoplasmic misfolded proteins and damaged organelles to maintain intracellular homeostasis. In cancer cells, autophagy is usually activated to benefit malignant cells to survive under adverse microenvironmental conditions [26]. A large body of evidence suggests that cellular autophagy is elevated by chemotherapy and/or radiation therapy [27]. It is also believed that autophagy plays dual roles in multidrug resistance (MDR) in chemotherapy. Autophagy can be triggered as a survival mechanism to mediate MDR. So, the inhibition of autophagy can sensitize the resistant cancer cells and enhance the antitumor effects of chemotherapeutic agents. But too strong autophagy may also induce autophagic cell death, which differs from apoptosis [28,29]. Therefore, in the current study, we first investigated the changes in autophagy levels triggered by Apatinib and AsPs in AGS cell line. The microtubule-associated protein LC3 conjugation system acts in membrane elongation and autophagosome formation. LC3-II converted from LC3-I during the early stages of autophagy. In our study, we found that both Apatinib and AsPs induced cellular autophagy, as proven by the increased LC3-I/II conversion which is involved in biosynthesis of the autophagosome. Nowadays, it is believed that autophagy has prosurvival and pro-death functions in gastric cancer cells and a crosstalk exists between autophagy and apoptosis. Autophagy can play a prosurvival role against cancer by eliminating damaged organelles. Paradoxically, excessive autophagy can devote cancer cells to “autophagic cell death”. Thus, autophagy induced by metabolic and therapeutic stresses can have a pro-death or pro-survival role, which depends on the different cell types and the intracellular signaling environment [30].
used as adjuvant to increase chemosensitivity and improve antitumor effects of chemotherapy, leading to prolonged survival time of the patients. AsPs has been well recognized as an antitumor preparation and is able to induce cell apoptosis [20]. Moreover, AsPs injection integrated with cisplatin had significantly improved quality of life in patients with advanced non-small-cell lung cancer (NSCLC) compared with cisplatin alone [15]. A newly meta-analysis showed that AsPs + FOLFOX (5fluorouracil, leucovorin and oxaliplatin) was superior in reducing leucopenia and gastrointestinal reaction compared with FOLFOX alone in patients with gastric cancer [21]. So, we think the combination of Apatinib and AsPs is a reasonable regime for gastric cancer treatment in the future. In the current study, we found AsPs enhanced the inhibitory effects of Apatinib on AGS cell. It was demonstrated that AsPs inhibited cell proliferation in a dose-dependent manner. We also observed that AsPs at 200 μg/ml remarkably increased the inhibitory effects of Apatinib on cell proliferation, migration and invasion as compared to the use of Apatinib alone. MMP-9 protein is closely associated with cell invasion and tumor metastasis, and its high expression could result in poor prognosis [22,23]. Western blotting results showed MMP-9 expression was inhibited by Apatinib and the combination of Apatinib with AsPs inhibited its expression to greater extent. Based on our findings, we inferred that perhaps Apatinib blocked cell migration and invasion by inhibiting MMP-9 expression and the addition of AsPs inhibited its expression more significantly. Moreover, flow cytometry results revealed that Apatinib-induced apoptosis was increased by the administration of AsPs to greated extent. Our results are consistent with the conclusion drawn by another study which revealed that AsPs could enhance the chemosensitivity of tumor cells to the majority of known chemotherapies in vitro, including Adriamycin, 5-Fluorouracil, cisplatin and so forth [24]. To clarify the underlying mechanisms of the synergistic effect of AsPs on Apatinib, p-AKT levels were determined by western blotting. AKT is a key apoptosis-related protein that promotes cell survival via inhibition of apoptosis. Over activation of p-AKT in 180
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Fig. 3. AsPs enhanced the inhibition of cell migration and invasion suppressed by Apatinib. AGS migration (A-B) was measured by wound-healing analysis for 0 and 24 h (original magnification × 40) and cell invasion (C-D) by Transwell analysis for 24 h (original magnification × 100). Cells were treated with control, Apatinib 20 μg/ml, AsPs 200 μg/ml and Apatinib 20 μg/ml + AsPs 200 μg/ml. (E) MMP-9 protein expression was determined by western blotting after 24 h. β-actin was used as the internal control. *P < .05, **P < .01, *** P < .001 vs control; #P < .05 vs Apatinib.
studied. To determine the relationship between the elevated autophagy and cell apoptosis, we utilized the autophagy inhibitor 3-MA to block cellular autophagy and found that the percentage of apoptotic cells increased significantly in Apatinib and Apatinib + sPs-treated group following 3-MA administration. Furthermore, the administration of 3-MA
Moreover, Autophagy and apoptosis could govern cell fate in independent or cooperative routes. On the other hand, Autophagy may protect cancer cells from death in stress conditions [31]. The specific mechanisms underlying the pro-survival and pro-death function of autophagy remains largely unknown and the interplay between autophagy and apoptosis remains controversial and needs to be further 181
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Fig. 4. AsPs increased the inhibition of p-AKT expression. AGS was treated with 0 (Control), AP (Apatinib 20 μg/ml), AsPs (200 μg/ml) and AP + AsPs (Apatinib 20 μg/ml + AsPs 200 μg/ml) for 24 h (A). Apatinib and AsPs induced cellular autophagy. The autophagy-related protein (LC3) from different groups was determined by western blotting. AGS was incubated with Apatinib at 0, 10 μg/ml, 20 μg/ml and 30 μg/ml and AsPs at 0, 200 μg/ml, 400 μg/ml and 600 μg/ml for 24 h (B-C). Cells were treated with 0 (Control), AP (Apatinib 20 μg/ml), AsPs (200 μg/ml) and AP + AsPs (Apatinib 20 μg/ ml + AsPs 200 μg/ml) for 24 h (D).
Fig. 5. Autophagy inhibitor 3-MA inhibited cell proliferation and increased apoptosis further. Cells were pre-treated with autophagy inhibitor 3-MA (300 μg/ml) for 1 h, and then incubated by Control, Apatinib (20 μg/ml), AsPs (200 μg/ml) and Apatinib (20 μg/ml)+AsPs (200 μg/ml) for 24 h. Cell proliferation was measured by MTS (A). Annexin V-FITC and PI staining assay were used to detected cell apoptosis, and the apoptosis percentage was analyzed by flow cytometry (B-C). *P < .05, Apatinib vs Apatinib + 3-MA, #P < .05, Apatinib + AsPs vs Apatinib + AsPs + 3-MA.
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significantly reduced the cell proliferation in Apatinib and Apatinib + AsPs-treated group as detected by MTS assay. The above evidence indicated that the elevated autophagy induced by Apatinib protected cells from apoptosis, therefore in this case the elevated autophagy by Apatinib could have an adverse effect in chemotherapy. In another study, bevacizumab as VEGF monoclonal antibody also activated autophagy in colon cancer cell, and the addition of chloroquine (autophagy inhibitor) provided greater tumor control in concert with evidence of autophagy inhibition [32]. Apatinib was also found to induce autophagy in colon cancer cells [17]. In osteosarcoma cells, Apatinib inhibited cell growth and induced autophagy. Consistent with our study, inhibition of autophagy increased Apatinib-induced apoptosis in osteosarcoma cells [33]. Recently targeting autophagy holds great potential for cancer treatment [34], autophagy inhibition could enhance the chemotherapy of anti-cancer drugs in gastric cancer cells [35]. Meanwhile, inducing autophagic cell death could also overcome multidrug resistance [36]. Therefore, inhibition of autophagy might be a potential strategy for cancer treatment. Unraveling the complex molecular regulation and diverse roles of autophagy is of great importance to guide development of cancer therapy in the future.
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Candido, S.L. Abrams, L.S. Steelman, K. Lertpiriyapong, T.L. Fitzgerald, A.M. Martelli, et al., Roles of NGAL and MMP-9 in the tumor microenvironment and sensitivity to targeted therapy, Biochim. Biophys. Acta. 1863 (2016) 438–448. [23] Z. Yao, T. Yuan, H. Wang, S. Yao, Y. Zhao, Y. Liu, et al., MMP-2 together with MMP-9 overexpression correlated with lymph node metastasis and poor prognosis in early gastric carcinoma, Tumour Biol. 39 (2017) pp. 1010428317700411. [24] Q.E. Tian, J.H. De, M. Yan, H.L. Cai, Q.Y. Tan, W.Y. Zhang, Effects of Astragalus polysaccharides on P-glycoprotein efflux pump function and protein expression in H22 hepatoma cells in vitro, BMC Complement. Altern. Med. 12 (2012) 94. [25] V. Asati, D.K. Mahapatra, S.K. Bharti, PI3K/Akt/ mTOR and Ras/Raf/ MEK/ERK signaling pathways inhibitors as anticancer agents: structural and pharmacological perspectives, Eur. J. Med. Chem. 109 (2016) 314–341. [26] H. Maes, N. Rubio, A.D. Garg, P. Agostinis, Autophagy: shaping the tumor microenvironment and therapeutic response, Trends. Mol. Med. 19 (2013) 428–446. [27] L. Galluzzi, J.M. Bravo-San Pedro, S. Demaria, S.C. Formenti, G. Kroemer, Activating autophagy to potentiate immunogenic chemotherapy and radiation therapy, Nat. Rev. Clin. Oncol. 14 (2017) 247–258. [28] S. Aveic, G.P. Tonini, Resistance to receptor tyrosine kinase inhibitors in solid tumors: can we improve the cancer fighting strategy by blocking autophagy? Cancer Cell. Int. 16 (2016) 62. [29] Y.J. Li, Y.H. Lei, N. Yao, C.R. Wang, N. Hu, W.C. Ye, et al., Chen. autophagy and multidrug resistance in cancer, Chin. J. Cancer 36 (2017) 52. [30] H.R. Qian, Y. Yang, Functional role of autophagy in gastric cancer, Oncotarget. 7 (2016) 17641–17651. [31] A. El-Khattouti, D. Selimovic, Y. Haikel, M. Hassan, Crosstalk between apoptosis and autophagy: molecular mechanisms and therapeutic strategies in cancer, J. Cell Death 6 (2013) 37–55. [32] M. Selvakumaran, R.K. Amaravadi, I.A. Vasilevskaya, P.J. O’Dwyer, Autophagy inhibition sensitizes colon cancer cells to antiangiogenic and cytotoxic therapy, Clin. Cancer Res. 19 (2013) 2995–3007. [33] K. Liu, T. Ren, Y. Huang, K. Sun, X. Bao, S. Wang, et al., Apatinib promotes autophagy and apoptosis through VEGFR2/STAT3/BCL-2 signaling in osteosarcoma, Cell Death Dis. 8 (2017) e3015. [34] D.C. Rubinsztein, P. Codogno, B. Levine, Autophagy modulation as a potential therapeutic target for diverse diseases, Nat. Rev. Drugs Discov. 11 (2012) 709–730. [35] F. Du, Y. Feng, J. Fang, M. Yang, MicroRNA-143 enhances chemosensitivity of Quercetin through autophagy inhibition via target GABARAPL1 in gastric cancer cells, Biomed. Pharmacother. 74 (2015) 169–177. [36] S.K. Bhutia, S. Mukhopadhyay, N. Sinha, D.N. Das, P.K. Panda, S.K. Patra, et al., Autophagy: cancer’s friend or foe? Adv. Cancer. Res. 118 (2013) 61–95.
5. Conclusion Our study showed AsPs enhanced antitumor effects of Apatinib in AGS cell line by inhibiting AKT signaling pathway. We discovered both Apatinib and AsPs elevated celluar autophagy. The application of autophagy inhibitor decreased cell viability and increased cell apoptosis. Thus, the combination of Apatinib and AsPs might be a potential therapeutic mixture in gastric cancer, and autophagy inhibitor might enhance the anti-tumor effects of Apatinib. However, the clinical application of Apatinib and AsPs is hampered by the lack of clinical trial evaluation and should be studied further in randomized controlled clinical trials. Funding This work was supported by Grants No. 7172081 from the Beijing Natural Science Foundation (to Bangwei Cao) and Grants No JJ2016-16 from the Traditional Chinese Medicine Science and Technology Development Fund Project of Beijing (to Bangwei Cao) and Grants No. QML20150107 from Administration of Hospitals Youth Programme (to Jing Wang). Disclosure None. Conflict of interest statement The authors have no conflict of interest. Acknowledgements We specially thank Dr. Zhaoyu Zhong for revising the manuscript. References [1] A. Jemal, F. Bray, M.M. Center, J. Ferlay, E. Ward, D. Forman, Global cancer statistics, CA. Cancer. J. Clin. 61 (2011) 69–90. [2] G. Group, K. Oba, X. Paoletti, Y.J. Bang, H. Bleiberg, T. Burzykowski, et al., Role of chemotherapy for advanced/recurrent gastric cancer: an individual – patient-data metaanalysis, Eur. J. Cancer 49 (2013) 1565–1577. [3] E. Lieto, F. Ferraraccio, M. Orditura, P. Castellano, A.L. Mura, M. Pinto, et al., Expression of vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) is an independent prognostic indicator of worse outcome in gastric cancer patients, Ann. Surg. Oncol. 15 (2007) 69–79. [4] R.J. Roskoski, Vascular endothelial growth factor (VEGF) and VEGF receptor inhibitors in the treatment of renal cell carcinomas, Pharmacol. Res. 120 (2017) 116–132.
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Astragalus polysaccharide enhanced antitumor effects of Apatinib in gastric cancer AGS cells by inhibiting AKT signalling pathway
T
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Jun Wua, Junxian Yub, Jing Wanga, Chenguang Zhangc, Kun Shanga, Xiaojun Yaod, , ⁎ Bangwei Caoa, a
Cancer Center, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China Department of Pharmacy, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China c Department of Biochemistry and Molecular biology, Capital Medical University, Beijing 100069, China d State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau, 999078, China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Gastric cancer Apatinib AsPs AKT
Apatinib has been proved effective in the treatment of advanced gastric cancer. Traditional Chinese medicine is often considered as adjuvants which could increase the effects and counteract the side effects of chemotherapy. The present study aims to explore the antitumor effects of Astragalus polysaccharide (AsPs) in combination with Apatinib in gastric cancer AGS cells. Our results demonstrated that the expression of VEGFR-2 was observed in human gastric cancer line AGS. Both Apatinib and AsPs could significantly inhibit the proliferation of AGS cells in a dose-dependent manner and Apatinib in combination with AsPs showed enhanced inhibitory effects on cell proliferation, migration and invasion compared with Apatinib monotherapy. Moreover, there was a remarkable increase in apoptosis following Apatinib treatment which could be enhanced by the addition of AsPs. Western blotting showed that the combination of Apatinib and AsPs could inhibit the expression of phosphorylated AKT (p-AKT) and MMP-9 expression. In addition, both Apatinib alone and Apatinib in combination with AsPs induced celluar autophagy which could be attenuated by the autophagy inhibitor 3-MA. The suppression of autophagy leaded to further apoptosis induction and cell proliferation suppression. In conclusion, the current study showed AsPs enhanced antitumor effects of Apatinib on AGS cells by the mechanism which includes inhibition of AKT signaling pathway. Apatinib-induced autophagy could be attenuated by 3-MA, which subsequently increased the apoptosis rate. On the basis of our study, the combination of Apatinib and AsPs could be considered as a potential candidate in the gastric cancer treatment.
1. Introduction Gastric cancer is one of the most malignant tumors worldwide and approximately half of the patients with gastric cancer are in China [1]. Despite a worldwide decline in incidence over the past few decades, most of the patients are still diagnosed at the advanced stage of illness due to the lack of early clinical symptoms and sensitive markers. Although many efforts have been made in gastric cancer treatment, the prognosis is still very poor with a median overall survival time (OS) less than 1year [2]. In the recent years, molecular-targeted therapy has become the research hotspot in cancer treatment. A number of targeted agents have shown effectiveness in cancer patients. Angiogenesis plays very important role in tumor development and metastasis. Vascular endothelial growth factor (VEGF) which binds to the high-affinity
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receptors (VEGFR) is a key mediator of angiogenesis [3]. A few angiogenesis inhibitors have shown efficacy in human tumors, such as in lung, breast and colorectal cancers [4–6]. Targeting the VEGF/VEGFR pathway is a viable alternative for gastric cancer treatment due to the high levels of VEGF/VEGFR expression which is associated with poor prognosis [7]. A systematic review and meta-analysis showed angiogenesis inhibitor-containing regimens were superior to routine regimens without angiogenesis inhibitor in patients with advanced gastric cancer [8]. Apatinib, a small-molecule tyrosine kinase inhibitor (TKI), specifically targets the intracellular ATP-binding domain of VEGFR-2, and subsequently inhibits angiogenesis by blocking the downstream signal transduction [9]. Apatinib was demonstrated effective in improving OS and progression-free survival (PFS) for patients with chemotherapy-
Corresponding author at: Cancer Center, Beijing Friendship Hospital, Capital Medical University, 95 Yongan Road, Xicheng District, Beijing 100050, China. Corresponding author. E-mail addresses: [email protected] (X. Yao), [email protected] (B. Cao).
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https://doi.org/10.1016/j.biopha.2018.01.140 Received 21 October 2017; Received in revised form 2 January 2018; Accepted 28 January 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
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of 5 μl PI solution to each group. Percentage of cell in the early and late stage of apoptosis was determined by flow cytometry.
refractory advanced or metastatic gastric cancers in the clinical phase III trial [10]. Chinese traditional medicine was used to treat a wide range of human diseases including cancers. It usually serves as adjuvants to increase the effects and/or counteract the side effects of chemotherapy [11,12]. Astragalus polysaccharide (AsPs), the active component extracted from Astragali Radix which is the root of Astragalus membranaceus Bunge [13], was reported to have antitumor effects by improving immunity, inducing apoptosis and inhibiting tumor growth. Moreover it was also shown to reduce the toxicity of chemotherapeutics [14,15]. In the present study, we investigated the enhanced antitumor effects of AsPs combined with Apatinib in gastric cancer cell. The alterations of cellular autophagy affected by Apatinib and AsPs were also explored.
2.4. Migration assay Cell migration was assessed by wound healing assay. AGS cells were seeded into 12-well plates. To create a “scratch’’, cell monolayers in each well were scratched in a straight line with a 200 μl-pipette tip when the cell confluency reached about 90%. We removed the debris and smoothed the edge of the scratch by washing the culture with PBS twice and then replaced with 1 ml DMEM containing different reagents without FBS for 24 h. The concentration of Apatinib and AsPs was 20 μg/ml and 200 μg/ml, respectively. It is important to create scratches of approximately similar width in the variously treated cells. The scratch lines were inspected at 0 and 24 h. Image fields were captured by using an inverted phase contrast microscope equipped with a digital camera (Olympus, Tokyo, Japan). By comparing the images from time 0 h to 24 h, we were able to obtain/calculated the distance of each scratch closure on the basis of the following formula: Cell motility = (distance 24 h - distance 0 h)/distance 0 h. The images acquired for each sample were further analyzed quantitatively by ImageJ Plus. Each experiment was performed in triplicate.
2. Materials and methods 2.1. Cell culture and reagents HUVEC (human umbilical vein endothelial cell) and human gastric cancer cell line AGS were acquired from the department of experimental research center in Beijing Friendship Hospital (Beijing, China). AGS and HUVEC were cultured in Dulbecco Modified Eagle Medium (DMEM, Corning, Virginia, VA, USA) supplemented with 10% FBS (fetal bovine serum, Biological Industries, Israel) and 1% penicillin/ streptomycin (KeyGen, Nanjing, China) in a 5% CO2 atmosphere at 37 °C. Apatinib was a gift of Jiangsu Hengrui medicine Co, Ltd (Jiangsu, China). AsPs was purchased from Nanjing Zelang Biotech Ltd (Nanjing, China). Both Apatinib and AsPs were dissolved by Dimethyl sulfoxide (DMSO), and both of them were prepared freshly prior to each test. 3Methyladenine (3-MA) was purchased from Sigma-Aldrich (St. Louis, MO, USA).
2.5. Invasion assay Transwell assay was performed to determine cell invasion. The coculture chambers (24-wells, 8-μm pore size, Costar, Corning Inc., ME, USA) were used in this section. In brief, prechilled upper inserts were coated with 60 μl Matrigel (Corning, USA) per well (1:20 in dilution) and then placed at 37 °C. The dilution agent was prechilled DMEM without FBS. After 6 h, upper inserts were hydrated by DMEM without FBS for 1 h at room temperature prior to addition of AGS cells. 3 × 104 cells per well suspended in 200 μL serum-free DMEM medium were added into the upper chamber and 600 μL DMEM medium containing 10% FBS was placed in the lower chamber. The concentration of Apatinib and AsPs was 20 μg/ml and 200 μg/ml. After 24 h incubation at 37 °C in a 5% CO2 atmosphere, the invaded cells left at the bottom of chambers were fixed in 4% paraformaldehyde for 25 min and then stained with 0.5% crystal violet (Macklin, Shanghai, China) for 15 min at room temperature. The cells at the upper layer were carefully removed with a cotton swab while the invaded cells at the lower layer were counted after air-drying. The crystal violet-positive cells were counted in 6 random visual fields for each chamber using an inverted phase contrast microscope (Olympus, Japan).
2.2. Cell proliferation assay MTS (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxypheny)-2(4-sulfophenyl) -2H- tetrazolium, innersalt), a CellTiter-96 AqueousOne Solution Cell Proliferation Assay Kit (Promega, Madison, WI, USA) was performed to detect cell proliferation. The cells were cultured in 96-well plates at a density of 8 × 103 cells per well, and then treated with DMEM containing reagents in different concentrations according to the experimental design when cells adhered overnight. Cells were treated with Apatinib in concentrations ranging from 0 to 30 μg/ml and AsPs from 0 to 600 μg/ml for 24 h. MTS solution (10 μl/well) was added to each experimental well and cultured for 1 h in a 5% CO2 atmosphere at 37 °C. The absorbance was measured on The ELx808™ Absorbance Microplate Reader (BioTek, Winooski, VT, USA) at the wave length of 490 nm. The viability of cell proliferation is directly proportional to A490 value. The concentration to inhibit cell proliferation by 50% (IC50) was calculated by using a linear regression equation. Triplicate experiments were performed in parallel manner for each concentration point.
2.6. Western blotting AGS cells were treated with reagents at indicated concentrations after 24 h incubation in 6-cm2 plates. The whole proteins were lysed in RIPA buffer (Applygen, Beijing, China) supplemented with protease inhibitor cocktail (Amresco, Ohio, USA) and the concentrations were quantified with a bicinchoninic acid (BCA) protein assay kit (Merck, Darmstadt, Germany). Protein samples were mixed with 5 × loading buffer (Applygen, Beijing, China) and heated at 95 °C for 10 min. An equal amount of 30 μg protein was separated by SDS-PAGE gel (sodium dodecyl sulfate- polyacrylamide gel electrophoresis) and then transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked with blocking buffer TBST (Tris-buffered saline and 0.2% Tween) containing 1.5% BSA (Bovine Serum Albumin) for 2 h at room temperature, and then incubated overnight with specific primary antibodies at 4 °C. Then the membranes were washed three times by TBST and incubated with HRP (horseradish peroxidase)-conjugated secondary antibody for 1 h at room temperature. LC3 rabbit anti-human monoclonal antibody (1:1000) and AKT rabbit anti-human monoclonal antibody (1:1000) were purchased from Cell Signaling Technology (Beverly, MA, USA). VEGFR-2 (1:1000) rabbit anti-human
2.3. Apoptosis assay Annexin V-FITC and propidium iodide (PI) staining assay were used to determine cell apoptosis by an apoptosis detecting kit (KeyGen, Nanjing, China) according to the manufacturer's instructions. AGS cells were plated and grown in 6-well plates in cultured standard growth conditions until they reached 70% confluency and then exposed to various treatments for 24 h. The concentration of Apatinib and AsPs was 20 μg/ml and 200 μg/ml, respectively. After being harvested by trypsin solution without EDTA (Ethylene Diamine Tetraacetic Acid), cells were washed with pre-chilled PBS (phosphate buffer saline) twice and then centrifuged at 2000 rpm/min for 5 min. The cell pellets were resuspended in binding buffer of 300 μl and incubated with 5 μl Annexin V-FITC on ice in the darkness for 15 min followed by addition 177
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3.4. AsPs enhanced the inhibition of cell migration and invasion caused by Apatinib
monoclonal antibody was purchased from Abcam (Cambridge, UK). MMP-9 (1:100) mouse anti-human monoclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); CST, USA). PAKT rabbit anti-human polyclonal antibody (1:500) was purchased from Abclonal (Wuhan, China). β-Actin rabbit anti-human monoclonal antibody (1:3000) as the interval control was obtained from Abcam (Cambridge, UK). The goat anti-rabbit and anti-mouse IgG conjugated to HRP antibody (1:5000; Santa Cruz Biotechnology Inc.) were used as the secondary antibody. Detection for visualization was performed by an enhanced chemiluminescence detection system (ECL, Millipore, USA).
Wound-healing and Transwell assay were conducted to determine the effects of Apatinib and AsPs on cell migration and invasion, respectively. Wound-healing assay results showed the Apatinib-treated group effectively inhibited cell migration (P < .05, Fig. 3A-B), while the AsPs-treated group did not affect the scratch closure significantly compared with the control group (P > .05). We also found Apatinib combined with AsPs inhibited cell migration to greater extent in comparison with Apatinib-treated group as shown in Fig. 3A-B (P < .05). Meanwhile, consistent with the wound-healing results, Transwell assay showed Apatinib decreased the invasive cell numbers significantly compared with the control group after statistical analysis as shown in Fig. 3C-D (P < .01). When Apatinib was combined with AsPs, inhibition in cell invasion to greater extent was observed compared with the Apatinib-treated group (P < .05). To explore the mechanisms behind the results above, MMP-9 protein expression was detected by western blotting. Results show MMP-9 expression was inhibited significantly by Apatinib after 24 h treatment as shown in Fig. 3E (P < .05), and the Apatinib + AsPs-treated group inhibited the expression of MMP-9 to greater extent. However, AsPs alone did not significantly inhibit MMP-9 expression as compared with the control group (P > .05, Fig. 3E).
2.7. Statistical analysis The data presented in this study were expressed as mean ± D. Statistical analysis and graphs were performed by GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA). Differences between groups were analysed using one-way analysis of variance (ANOVA). Values of P < .05 were considered statistically significant. 3. Results 3.1. VEGFR2 expression in AGS cell line
3.5. AsPs enhanced the inhibition of p-AKT expression
Firstly, AGS cell line was evaluated for VEGFR-2 expression by western blotting and HUVEC was used as the positive control for VEGFR-2 expression. Cells were cultured in 6-cm2 plates and the procedures of western blotting were described in the Materials and methods section. As shown in Fig. 1A, VEGFR-2 expression was detected in AGS cell line.
Western blotting was carried out to determine the effects of Apatinib and AsPs on p-AKT, which is the downstream effector molecule in the VEGFR-2 signaling pathway. After 24 h incubation, we found Apatinib decreased the level of p-AKT expression, and Apatinib combined with AsPs inhibited p-AKT expression in even greater extent (P < .05, Fig. 4A). However, compared with the control group, AsPs used at 200 μg/ml alone did not change p-AKT expression significantly (P > .05).
3.2. Apatinib and AsPs inhibited cell proliferation MTS results showed that with the increase of reagent concentration, either Apatinib or AsPs reduced A490 value as shown in Fig. 1B-C. As compared with the control group, Apatinib at 20 μg/ml and AsPs at 500 μg/ml displayed significant inhibiting effects on proliferation in AGS (P < .001). The IC50 values of Apatinib and AsPs were 26.3 μg/ml and 660.2 μg/ml, respectively. To clarify whether AsPs could increase the inhibitory effects of Apatinib on cell proliferation, we chose Apatinib at 20 μg/ml combined with AsPs at 50, 100, 200 and 400 μg/ml. Results revealed that Apatinib in combination with AsPs at 200 μg/ml produced stronger inhibition on cell proliferation compared with Apatinib used alone as shown in Fig. 1D (P < .01). Moreover, Apatinib at 20 μg/ml in combination with AsPs at 200 μg/ml decreased the IC50 values to 239.9 μg/ml. However, AsPs alone at 200 μg/ml did not display inhibitory effects (P > .05, Fig. 1C). Therefore, we chose the combination of Apatinib at 20 μg/ml and AsPs at 200 μg/ml to explore the mechanisms of AsPs exerting the enhanced antitumor effects in the subsequent experiment.
3.6. Apatinib and AsPs induced cellular autophagy To explore whether Apatinib and AsPs could change cellular autophagy level, LC3, a specific marker of autophagy was detected by western blotting. Cells were incubated with various concentration of Apatinib (0, 10, 20, 30 μg/ml) and AsPs (0, 200, 400, 600 μg/ml) for 24 h. Results showed either Apatinib or AsPs increased LC3 expression in a dose-dependent manner (P < .05, Fig. 4B,C). Apatinib at 20 μg/ml combined with AsPs at 200 μg/ml significantly increased LC3 expression compared with the Apatinib-treated group (Fig. 4D, P < .05). Therefore, the results indicate that both Apatinib and AsPs induced cellular autophagy. 3.7. Autophagy inhibitor 3-MA inhibited cell proliferation and increased apoptosis more significantly To determine the potential role of the increased autophagy induced by Apatinib and AsPs on cell proliferation, cells were pre-treated with autophagy inhibitor 3-MA (300 μg/ml) for 1 h, followed by various treatments for 24 h. The concentration of Apatinib and AsPs was 20 μg/ ml and 200 μg/ml, respectively. MTS assay showed Apatinib and Apatinib + AsPs in combination with 3-MA significantly inhibited cell proliferation as compared to the single use of above compounds (Fig. 5A, P < 0.05). Furthermore, AsPs + 3-MA and 3-MA monotherapy showed no obvious effect on cell proliferation (Fig. 5A, P > .05). To clarify the effects of 3-MA on cell apoptosis, Annexin VFITC and PI staining assay were used to detect cell apoptosis, and the percentage of apoptosis was also obtained by flow cytometry. As shown in Fig. 5B-C, we found that Apatinib + 3-MA (17.7 ± 3.2%) and Apatinib + AsPs + 3-MA (23.2 ± 3.7%) significantly increased cell apoptosis compared with them used singly (Apatinib: 13.3 ± 2.9%;
3.3. AsPs combined with Apatinib increased cell apoptosis To reveal the effects of Apatinib and AsPs on cell apoptosis, Annexin V and PI staining assay were performed to detect apoptosis rate of AGS cells treated with various reagents for 24 h and then the apoptosis percentage was analyzed by flow cytometry. As shown in Fig. 2, the percentage of apoptotic cells increased from 6.9 ± 3.1% in the control group to 12.5 ± 2.9% in the Apatinib-treated group (P < .01). We observed the Apatinib + AsPs-treated group significantly increased apoptosis percentage compared with the Apatinib-treated group (19.7 ± 3.4%:12.5 ± 2.9%, P < .01). However, compared with the control group, AsPs at 200 μg/ml in a single use did not enhance apoptosis percentage (5.8 ± 2.6%:6.9 ± 3.1%, P > .05). Therefore, we concluded that Apatinib induced cell apoptosis and AsPs enhanced apoptosis induced by Apatinib. 178
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Fig. 1. Expression of VEGFR-2 in AGS. HUVEC was used as the positive control for VEGFR-2, Western blotting showed VEGFR-2 was detected in AGS (A). AsPs enhanced the inhibitory effects of Apatinib on cell proliferation. MTS showed either Apatinib or AsPs reduced A490 value in a dose-dependent manner after 24 h treatment (B-C). Apatinib at 20 μg/ml in combination with AsPs at 200 μg/ml showed stronger inhibition on cell proliferation compared with the single use of Apatinib group (D). *P < .05, **P < .01, ***P < .001 vs control; ## P < .01, ###P < .001 vs Apatinib 20 μg/ml.
Apatinib + AsPs: 16.8 ± 3.3%; P < .05), (8.9 ± 2.6%) did not increase cell apoptosis to pared with AsPs alone (8.1 ± 2.5%; P > .05). control group (5.6 ± 2.3%), 3-MA alone (7.1 ± cell apoptosis (P > .05).
Apatinib, a novel oral antiangiogenic agent, was approved as a third-line treatment for gastric cancer patients. In our study, Apatinib was proved effective in inhibiting cell proliferation, migration and invasion of AGS cell line. In addition it also induced cell apoptosis. Our findings are consistent with the results of a new study that showed Apatinib inhibited cell viability, migration and invasion capabilities and increased apoptosis percentage in colon cancer cells [17]. However, drug resistance remains a major problem in tumor therapy including antiangiogenic therapies [18]. Long-term VEGF/VEGFR2 inhibition like bevacizumab (VEGF monoclonal antibody) was found to lead to the upregulation of other tumor-derived angiogenic factors, including platelet-derived growth factor (PDGF), angiopoietin (Ang), hepatocyte growth factor (HGF), and epidermal growth factor (EGF), which contributed to the mechanism of acquired resistance [19]. So, we infer Apatinib probably face the same difficulty in the years to come. At present, due to its little toxicity, Chinese traditional medicine is usually
but AsPs + 3-MA greater extent comCompared with the 2.1%) did not affect
4. Discussion According to the latest cancer statistics in China, gastric cancer is identified as the second leading cause of cancer incidence and mortality, posing a huge threat to public health [16]. Conventional chemotherapy or postoperative adjuvant chemotherapy is beneficial to gastric cancer patients, but some patients could not bear it because of its multiple adverse effects, resulting in discontinuation of treatment. In recent years, a series of antiangiogenic agents have been developed and improved prognosis of gastric cancer patients in clinical trials [8]. 179
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Fig. 2. AsPs increased apoptosis induced by Apatinib in AGS. AGS exposed to control, Apatinib 20 μg/ml, AsPs 200 μg/ml and Apatinib 20 μg/ml + AsPs 200 μg/ml after 24 h followed by Annexin V-FITC and propidium iodide staining and apoptosis percentage was detected by flow cytometry. **P < .01, ***P < .001 vs control; ##P < .01 vs Apatinib.
AKT signaling pathway could prevent cell apoptosis and enhance cell proliferation and migration in human cancers [25]. We found that the p-AKT expression was inhibited by Apatinib. This could be additionally suppressed by the administration of AsPs. Based on our findings, we believe that AsPs potentiates the antitumor effects of Apatinib on AGS cells by blocking AKT signaling pathway. Autophagy is the fundamental process through which cells utilize lysosomal hydrolases to degrade cytoplasmic misfolded proteins and damaged organelles to maintain intracellular homeostasis. In cancer cells, autophagy is usually activated to benefit malignant cells to survive under adverse microenvironmental conditions [26]. A large body of evidence suggests that cellular autophagy is elevated by chemotherapy and/or radiation therapy [27]. It is also believed that autophagy plays dual roles in multidrug resistance (MDR) in chemotherapy. Autophagy can be triggered as a survival mechanism to mediate MDR. So, the inhibition of autophagy can sensitize the resistant cancer cells and enhance the antitumor effects of chemotherapeutic agents. But too strong autophagy may also induce autophagic cell death, which differs from apoptosis [28,29]. Therefore, in the current study, we first investigated the changes in autophagy levels triggered by Apatinib and AsPs in AGS cell line. The microtubule-associated protein LC3 conjugation system acts in membrane elongation and autophagosome formation. LC3-II converted from LC3-I during the early stages of autophagy. In our study, we found that both Apatinib and AsPs induced cellular autophagy, as proven by the increased LC3-I/II conversion which is involved in biosynthesis of the autophagosome. Nowadays, it is believed that autophagy has prosurvival and pro-death functions in gastric cancer cells and a crosstalk exists between autophagy and apoptosis. Autophagy can play a prosurvival role against cancer by eliminating damaged organelles. Paradoxically, excessive autophagy can devote cancer cells to “autophagic cell death”. Thus, autophagy induced by metabolic and therapeutic stresses can have a pro-death or pro-survival role, which depends on the different cell types and the intracellular signaling environment [30].
used as adjuvant to increase chemosensitivity and improve antitumor effects of chemotherapy, leading to prolonged survival time of the patients. AsPs has been well recognized as an antitumor preparation and is able to induce cell apoptosis [20]. Moreover, AsPs injection integrated with cisplatin had significantly improved quality of life in patients with advanced non-small-cell lung cancer (NSCLC) compared with cisplatin alone [15]. A newly meta-analysis showed that AsPs + FOLFOX (5fluorouracil, leucovorin and oxaliplatin) was superior in reducing leucopenia and gastrointestinal reaction compared with FOLFOX alone in patients with gastric cancer [21]. So, we think the combination of Apatinib and AsPs is a reasonable regime for gastric cancer treatment in the future. In the current study, we found AsPs enhanced the inhibitory effects of Apatinib on AGS cell. It was demonstrated that AsPs inhibited cell proliferation in a dose-dependent manner. We also observed that AsPs at 200 μg/ml remarkably increased the inhibitory effects of Apatinib on cell proliferation, migration and invasion as compared to the use of Apatinib alone. MMP-9 protein is closely associated with cell invasion and tumor metastasis, and its high expression could result in poor prognosis [22,23]. Western blotting results showed MMP-9 expression was inhibited by Apatinib and the combination of Apatinib with AsPs inhibited its expression to greater extent. Based on our findings, we inferred that perhaps Apatinib blocked cell migration and invasion by inhibiting MMP-9 expression and the addition of AsPs inhibited its expression more significantly. Moreover, flow cytometry results revealed that Apatinib-induced apoptosis was increased by the administration of AsPs to greated extent. Our results are consistent with the conclusion drawn by another study which revealed that AsPs could enhance the chemosensitivity of tumor cells to the majority of known chemotherapies in vitro, including Adriamycin, 5-Fluorouracil, cisplatin and so forth [24]. To clarify the underlying mechanisms of the synergistic effect of AsPs on Apatinib, p-AKT levels were determined by western blotting. AKT is a key apoptosis-related protein that promotes cell survival via inhibition of apoptosis. Over activation of p-AKT in 180
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Fig. 3. AsPs enhanced the inhibition of cell migration and invasion suppressed by Apatinib. AGS migration (A-B) was measured by wound-healing analysis for 0 and 24 h (original magnification × 40) and cell invasion (C-D) by Transwell analysis for 24 h (original magnification × 100). Cells were treated with control, Apatinib 20 μg/ml, AsPs 200 μg/ml and Apatinib 20 μg/ml + AsPs 200 μg/ml. (E) MMP-9 protein expression was determined by western blotting after 24 h. β-actin was used as the internal control. *P < .05, **P < .01, *** P < .001 vs control; #P < .05 vs Apatinib.
studied. To determine the relationship between the elevated autophagy and cell apoptosis, we utilized the autophagy inhibitor 3-MA to block cellular autophagy and found that the percentage of apoptotic cells increased significantly in Apatinib and Apatinib + sPs-treated group following 3-MA administration. Furthermore, the administration of 3-MA
Moreover, Autophagy and apoptosis could govern cell fate in independent or cooperative routes. On the other hand, Autophagy may protect cancer cells from death in stress conditions [31]. The specific mechanisms underlying the pro-survival and pro-death function of autophagy remains largely unknown and the interplay between autophagy and apoptosis remains controversial and needs to be further 181
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Fig. 4. AsPs increased the inhibition of p-AKT expression. AGS was treated with 0 (Control), AP (Apatinib 20 μg/ml), AsPs (200 μg/ml) and AP + AsPs (Apatinib 20 μg/ml + AsPs 200 μg/ml) for 24 h (A). Apatinib and AsPs induced cellular autophagy. The autophagy-related protein (LC3) from different groups was determined by western blotting. AGS was incubated with Apatinib at 0, 10 μg/ml, 20 μg/ml and 30 μg/ml and AsPs at 0, 200 μg/ml, 400 μg/ml and 600 μg/ml for 24 h (B-C). Cells were treated with 0 (Control), AP (Apatinib 20 μg/ml), AsPs (200 μg/ml) and AP + AsPs (Apatinib 20 μg/ ml + AsPs 200 μg/ml) for 24 h (D).
Fig. 5. Autophagy inhibitor 3-MA inhibited cell proliferation and increased apoptosis further. Cells were pre-treated with autophagy inhibitor 3-MA (300 μg/ml) for 1 h, and then incubated by Control, Apatinib (20 μg/ml), AsPs (200 μg/ml) and Apatinib (20 μg/ml)+AsPs (200 μg/ml) for 24 h. Cell proliferation was measured by MTS (A). Annexin V-FITC and PI staining assay were used to detected cell apoptosis, and the apoptosis percentage was analyzed by flow cytometry (B-C). *P < .05, Apatinib vs Apatinib + 3-MA, #P < .05, Apatinib + AsPs vs Apatinib + AsPs + 3-MA.
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significantly reduced the cell proliferation in Apatinib and Apatinib + AsPs-treated group as detected by MTS assay. The above evidence indicated that the elevated autophagy induced by Apatinib protected cells from apoptosis, therefore in this case the elevated autophagy by Apatinib could have an adverse effect in chemotherapy. In another study, bevacizumab as VEGF monoclonal antibody also activated autophagy in colon cancer cell, and the addition of chloroquine (autophagy inhibitor) provided greater tumor control in concert with evidence of autophagy inhibition [32]. Apatinib was also found to induce autophagy in colon cancer cells [17]. In osteosarcoma cells, Apatinib inhibited cell growth and induced autophagy. Consistent with our study, inhibition of autophagy increased Apatinib-induced apoptosis in osteosarcoma cells [33]. Recently targeting autophagy holds great potential for cancer treatment [34], autophagy inhibition could enhance the chemotherapy of anti-cancer drugs in gastric cancer cells [35]. Meanwhile, inducing autophagic cell death could also overcome multidrug resistance [36]. Therefore, inhibition of autophagy might be a potential strategy for cancer treatment. Unraveling the complex molecular regulation and diverse roles of autophagy is of great importance to guide development of cancer therapy in the future.
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5. Conclusion Our study showed AsPs enhanced antitumor effects of Apatinib in AGS cell line by inhibiting AKT signaling pathway. We discovered both Apatinib and AsPs elevated celluar autophagy. The application of autophagy inhibitor decreased cell viability and increased cell apoptosis. Thus, the combination of Apatinib and AsPs might be a potential therapeutic mixture in gastric cancer, and autophagy inhibitor might enhance the anti-tumor effects of Apatinib. However, the clinical application of Apatinib and AsPs is hampered by the lack of clinical trial evaluation and should be studied further in randomized controlled clinical trials. Funding This work was supported by Grants No. 7172081 from the Beijing Natural Science Foundation (to Bangwei Cao) and Grants No JJ2016-16 from the Traditional Chinese Medicine Science and Technology Development Fund Project of Beijing (to Bangwei Cao) and Grants No. QML20150107 from Administration of Hospitals Youth Programme (to Jing Wang). Disclosure None. Conflict of interest statement The authors have no conflict of interest. Acknowledgements We specially thank Dr. Zhaoyu Zhong for revising the manuscript. References [1] A. Jemal, F. Bray, M.M. Center, J. Ferlay, E. Ward, D. Forman, Global cancer statistics, CA. Cancer. J. Clin. 61 (2011) 69–90. [2] G. Group, K. Oba, X. Paoletti, Y.J. Bang, H. Bleiberg, T. Burzykowski, et al., Role of chemotherapy for advanced/recurrent gastric cancer: an individual – patient-data metaanalysis, Eur. J. Cancer 49 (2013) 1565–1577. [3] E. Lieto, F. Ferraraccio, M. Orditura, P. Castellano, A.L. Mura, M. Pinto, et al., Expression of vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) is an independent prognostic indicator of worse outcome in gastric cancer patients, Ann. Surg. Oncol. 15 (2007) 69–79. [4] R.J. Roskoski, Vascular endothelial growth factor (VEGF) and VEGF receptor inhibitors in the treatment of renal cell carcinomas, Pharmacol. Res. 120 (2017) 116–132.
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