| Neuroblastoma (NB) is the most common extracranial solid tumor among children that arises in the peripheral sympathetic nervous system. It typically emerges in the adrenal medulla or paraspinal ganglia during embryogenesis and accounts for about10%of pediatric cancer-related deaths. The clinical manifestations of NB were diverse. Prognosis depends on the patients’ age, locations, region and biological characteristics of NB, but it is usually poor. The treatment of NB mainly includes surgery, chemotherapy, radiotherapy, biological treatment, and simple observation in some choosy cases. The strength of treatment is varying depending on the differences of high, medium and low risk cases. In recent years, the survival rate of children with NB has increased significantly, as methods of early diagnosis have improved, but patients with high-risk cases have a chance of5-year survival still below40%. Nowadays, chemotherapy still plays an important role in the treatment of NB. Because of the pain of chemotherapy to the patients and side effects, it is urgent to looking for novel adjuvant agents suitable for treatment of NB with few side effects to increase overall survivals.Cucurbitacin B (CuB) is tetracyclic triterpenes isolated from Cucurbitaceae. It has a broad range of pharmacological effects, such as anti-viral, anti-inflammatory, and anticancer activities. It has significant anti-inflammatory activity and is used traditionally to treat hepatitis. The anticancer activities of CuB obtain the most widespread attention. CuB could inhibit cell proliferation and induce apoptosis through different molecular mechanisms in different cancer cells, and therefore becomes a potent anticancer drug. As yet, whether CuB has anticancer effect in NB cells and the exact molecular mechanism are still not clearly defined. Therefore, the NB cell line SH-SY5Y cells cultured in vitro were used as the material. We firstly detected the effect of CuB on SH-SY5Y cells, and then systematically studied the molecular mechanism of the inhibition of CuB in SH-SY5Y cells. The main results of present study are as follows:1) CuB inhibited the proliferation of SH-SY5Y cells by inducing cell cycle arrest at G2/M phase. Effect of CuB on SH-SY5Y cells proliferation was examined by MTT assay. The results showed that CuB inhibited cell proliferation in a concentration-and time-dependent manner. The cell cycle distributions of SH-SY5Y cells with CuB treatment were detected by flow cytometry after PI staining and the results showed that as the concentration of CuB increasing, the number of cells in G2/M phase increases, indicating the G2/M phase arrest.2) CuB induced SH-SY5Y cells apoptosis, promoted ROS production, depolarization of mitochondrial membrane, and the increase of the intracellular Ca2+level. The apoptosis effect of CuB on SH-SY5Y cells was detected by Annexin V-FITC/PI double staining. Results showed that CuB induced early apoptosis in SH-SY5Y. The morphologic changes were observed by inverted microscope and fluorescence microscope after Hoechst33258staining. DCFH-DA、JC-1and Fluo-3AM were respectively used to detect the changes of ROS, mitochondrial membrane potential and the level of intracellular Ca2+. Results showed that after CuB treatment, the level of ROS and intracellular Ca2+were increased, and the mitochondrial membrane potential was reduced in a dose-dependent manner.3) CuB induced cell cycle arrest and apoptosis in SH-SY5Y cells through regulating the expression of proteins involved in G2/M progression and apoptosis. In addition, CuB also up-regulated p53and p21, and down-regulated proteins related to angiogenesis, metastasis and invasion. The expression of genes and proteins involved in G2/M progression and apoptosis were detected by RT-PCR and Western blot, respectively. Results showed that CuB down-regulated Cyclin B1, CDK1and Bcl-2at both transcriptional and translational levels in a concentration-dependent manner. In addition, the activities of caspase-3,9and the expression of p53and p21were also detected, and results showed that they were all promoted by CuB. Meanwhile, the results of Western blot revealed that CuB inhibited the expression of VEGF, MMP-2, MMP-9and COX-2, which respectively related to angiogenesis, metastasis and invasion.4) CuB inhibited cell proliferation and induced apoptosis by inhibiting the expression of proteins in JAK2/STAT3signaling pathway. JAK2/STAT3plays an important role in cell proliferation, differentiation, migration and apoptosis. Then we detected the expression of proteins of JAK2/STAT3after CuB treatment for24h. Results showed that CuB inhibited the expression of p-JAK2and p-STAT3, but the expression of JAK2and STAT3did not show any obvious changes. After that, inhibitor AG490and activator IL-6were used to examine the protein expression. Results showed that CuB significantly inhibited JAK2/STAT3signaling pathway. To investigate the relationship between JAK2/STAT3signaling pathway and cell proliferation and apoptosis, MTT and flow cytometry were respectively used to detect the cell proliferation and apoptosis after CuB, AG490and IL-6treatment in SH-SY5Y cells. Results indicated that CuB inhibited cell proliferation and apoptosis through inhibiting the activation of JAK2/STAT3signaling pathway.5) MAPKs was involved in the regulation of CuB inhibited cell proliferation and apoptosis in SH-SY5Y cells. The results of Western blot revealed that CuB inhibited the activation of ERK, promoted the activation of JNK and p38MAPK. Inhibitors (PD98059, SP600125and SB203580) were then used to evaluate that CuB regulated the activation of MAPKs. MTT and flow cytometry were used to detect the relationship between MAPKs and cell proliferation and apoptosis. Results indicated that MAPKs was related to the proliferation inhibition and apoptosis in CuB-treated SH-SY5Y cells.In conclusion, our study indicated that CuB inhibited cell proliferation and induced apoptosis in vitro in SH-SY5Y cells, and it could be a candidate drug for NB treatment. |
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