Error bars represent means SEM of = 3

Error bars represent means SEM of = 3. H1975 cells could be arrested at the G0/G1 phase after cordycepin treatment. The expression levels of apoptosis-related protein Caspase-3 and Bcl-2 and phosphorylated expression levels of EGFR, AKT and ERK1/2 were all decreased compared with the control group stimulated with EGF. However, the protein expression levels of proapoptotic protein Bax and cleaved caspase-3 were increased. These results implied that cordycepin could inhibit cell proliferation and induce apoptosis via the EGFR signaling pathway. Our results indicated that there was potential to seek a novel EGFR inhibitor from cordycepin and its chemical derivatives. in 1951, which exhibits a variety of clinical health effects including immunomodulatory, anticancer, antioxidant, anti-inflammatory and anti-microbial activities [13,14,15]. It was reported first that cordycepin could inhibit the biosynthesis of purine and participate in the synthesis of RNA and/or DNA to exert its biological activity [16]. With the development of cell biology and molecular biology, studies about cordycepin regulating cancer cell progress appear more and more frequently. Choi and co-workers [17] reported that cordycepin could induce human leukemia cell apoptosis through a signal involving a ROS (Reactive Oxygen Species) mediated caspase pathway. Kazuki Nakamura [18] proved that the antitumor effect of cordycepin on lung carcinoma cells was induced via the stimulation of the adenosine A3 receptor. Liou et al. [19] reported that cordycepin could suppress integrin/FAK signaling and the epithelial-mesenchymal transition in hepatocellular carcinoma. Lee and co-workers [20] suggested that cordycepin could inhibit the migration and invasion of LNCap human prostate carcinoma cells by inactivation of AKT, resulting in the down-regulation of the TJs (Tight Junctions) and MMPs (Matix Metalloproteinases). Chen and co-workers [21] investigated the effect of cordycepin on C6 glioma cells and showed that cordycepin could induce the apoptosis of C6 glioma cells via the adenosine 2A receptor-p53-caspase-7-PARP pathway. Though almost all the studies reported that cordycepin could inhibit cell proliferation and induce apoptosis, the concrete mechanisms were not identical. It is confirmed that cordycepin can be converted into 5 mono-, di- and tri-phosphates in vivo to inhibit the ML277 activity of enzymes and interfere a number of biochemical and molecular processes [22,23]. The role of EGFR inhibitors is exerted by competitively fitting to the ATP binding pocket of EGFR to prevent the signal transduction [24]. The chemical structure of tri-phosphated cordycepin is very similar to ATP. Therefore, it is predicted that EGFR may be confused by cordycepin through incorporating it in the ATP binding pocket to affect the normal function of EGFR (Figure 1). For the similarity of tri-phosphated cordycepin with ATP, it was predicted that cordycepin may confuse EGFR for the incorporation of cordycepin in the ATP pocket to affect the normal function of EGFR (Figure 1). Open in a separate window Figure 1 Chemical structure of cordycepin and ATP. Taken together, we hypothesize that cordycepin can interrupt EGFR signal transduction to inhibit lung cancer cell proliferation and induce apoptosis. In the present study, we extracted and purified cordycepin form and explored the anticancer effect and its mechanisms. Human lung cancer cell line H1975 was employed and the phosphorylation of EGFR and its downstream molecular proteins AKT and ERK1/2 were investigated to verify the inhibitory role of cordycepin. 2. Results and Discussion 2.1. Cordycepin Extraction and Purification To get enough cordycepin, was extracted in water and the extraction solution was purified by Amberlite 732 column chromatography (0.15 mol/L ammonia water) and recrystallization in H2O. The chemical structure of the isolated cordycepin was determined by 1H-NMR, 13C-NMR and MS. 1H-NMR (400 MHz, DMSO): 8.39 (s, 1H), 8.17 (s, 1H), 7.32 (s, 2H), 5.85 (d, = 2.5 Hz, 1H), 5.69 (d, = 4.0 Hz, 1H ), 5.25 (t, = 5.7 Hz, 1H), 4.62 (m, 1H), 4.38 (m, 1H ), 3.57C3.63 (m. 2H), 1.83C1.95 (m,.ESI-MS for C10H13N5O3 [M + H]+: calcd: 251.12, found: 252.12, mp 229~230 C. 2.2. protein Caspase-3 and Bcl-2 and phosphorylated manifestation levels of EGFR, AKT and ERK1/2 were all decreased compared with the control group stimulated with EGF. However, the protein expression levels of proapoptotic protein Bax and cleaved caspase-3 were increased. These results implied that cordycepin could inhibit cell proliferation and induce apoptosis via the EGFR signaling pathway. Our results indicated that there was potential to seek a novel EGFR inhibitor from cordycepin and its chemical derivatives. in 1951, which exhibits a variety of medical health effects including immunomodulatory, anticancer, antioxidant, anti-inflammatory and anti-microbial activities [13,14,15]. It was reported 1st that cordycepin could inhibit the biosynthesis of purine and participate in the synthesis of RNA and/or DNA to exert its biological activity [16]. With the development of cell biology and molecular biology, studies about cordycepin regulating malignancy cell progress appear more and more regularly. Choi and co-workers [17] reported that cordycepin could induce human being leukemia cell apoptosis through a signal including a ROS (Reactive Oxygen Varieties) mediated caspase pathway. Kazuki Nakamura [18] proved the antitumor effect of cordycepin on lung carcinoma cells was induced via the activation of the adenosine A3 receptor. Liou et al. [19] reported that cordycepin could suppress integrin/FAK signaling and the epithelial-mesenchymal transition in hepatocellular carcinoma. Lee and co-workers [20] suggested that cordycepin could inhibit the migration and invasion of LNCap human being prostate carcinoma cells by inactivation of AKT, resulting in the down-regulation of the TJs (Tight Junctions) and MMPs (Matix Metalloproteinases). Chen and co-workers [21] investigated the effect of cordycepin on C6 glioma cells and showed that cordycepin could induce the apoptosis of C6 glioma cells via the adenosine 2A receptor-p53-caspase-7-PARP pathway. Though almost all the studies reported that cordycepin could inhibit cell proliferation and induce apoptosis, the concrete ML277 mechanisms were not identical. It is confirmed that cordycepin can be converted into 5 mono-, di- and tri-phosphates in vivo to inhibit the activity of enzymes and interfere a number of biochemical and molecular processes [22,23]. The part of EGFR inhibitors is definitely exerted by competitively fitted to the ATP binding pocket of EGFR to prevent the signal transduction [24]. The chemical structure of tri-phosphated cordycepin is very much like ATP. Therefore, it is expected that EGFR may be puzzled by cordycepin through incorporating it in the ATP binding pocket to impact the normal function of EGFR (Number 1). For the similarity of tri-phosphated cordycepin with ATP, it was expected that cordycepin may confuse EGFR for the incorporation of cordycepin in the ATP pocket to impact the normal function of EGFR (Number 1). Open in a separate window Number 1 Chemical structure of cordycepin and ATP. Taken collectively, we hypothesize that cordycepin can interrupt EGFR transmission transduction to inhibit lung malignancy cell proliferation and induce apoptosis. In the present study, we extracted and purified cordycepin form and explored the anticancer effect and ML277 its mechanisms. Human lung malignancy cell collection H1975 was used and the phosphorylation of EGFR and its downstream molecular proteins AKT and ERK1/2 were investigated to verify the inhibitory part of cordycepin. 2. Results and Conversation 2.1. Cordycepin Extraction ML277 and Purification To get plenty of cordycepin, was extracted in water and the extraction answer was purified by Amberlite 732 column chromatography (0.15 mol/L ammonia water) and recrystallization in H2O. The chemical structure of the isolated cordycepin was determined by 1H-NMR, 13C-NMR and MS. 1H-NMR (400 MHz, DMSO): 8.39 (s, 1H), 8.17 (s, 1H), 7.32 (s, 2H), 5.85 (d, = 2.5 Hz, 1H), 5.69 (d, = 4.0 Hz, 1H ), 5.25 (t, = 5.7 Hz, 1H), 4.62 (m, 1H), 4.38 (m, 1H ), 3.57C3.63 (m. 2H), 1.83C1.95 (m, 2H); 13C-NMR (400 MHz, DMSO): 147.53, 151.59, 156.33, 138.91, 118.95, 90.95 , 81.01, 74.69, 62.98, 33.92. ESI-MS for C10H13N5O3 [M + H]+: calcd: 251.12, found: 252.12, mp 229~230 C. 2.2. Cordycepin Inhibits H1975 Cell Proliferation The effect of cordycepin on proliferation of H1975 cells was determined by MTT Rabbit polyclonal to Receptor Estrogen alpha.ER-alpha is a nuclear hormone receptor and transcription factor.Regulates gene expression and affects cellular proliferation and differentiation in target tissues.Two splice-variant isoforms have been described. assay. As demonstrated in Number 2, after treatment with numerous concentrations (0.1, 0.5, 1, 2, 5, 10, 20, 50 g/mL) of cordycepin for 24, 48, and 72 h, the proliferation of H1975 cells was inhibited moderately. The collection chart exhibited that cordycepin could inhibit the proliferation of the H1975 cells concentration dependently. Half-maximal inhibitory concentration (IC50) values were determined by SPSS. The IC50 value of 48 h (15.34 M) was higher than that of 72 h (10.96 M), but was lower than the IC50 value of 24 h (25.56 M). These results indicated that cordycepin could inhibit the proliferation of H1975 cells inside a dose-dependent manner. To elucidate ML277 the inhibitor potency of cordycepin, gefitinib, from your.