Dataset Information

COX?2 promotes epithelial?mesenchymal transition and migration in osteosarcoma MG?63 cells via PI3K/AKT/NF??B signaling.

ABSTRACT: The present study aimed to investigate the mechanism by which cyclooxygenase?2 (COX?2) promotes the metastasis of MG?63 osteosarcoma cells through the PI3K/AKT/NF??B pathway. To achieve this, a recombinant lentivirus containing the COX?2 gene was constructed in order to overexpress COX?2; a recombinant lentivirus containing a control sequence was also constructed. A Transwell chamber migration assay was performed to quantify the migration of the COX?2?transduced cells, and of cells treated with a COX?2 inhibitor (NS398) or a PI3K inhibitor (LY294002). Immunofluorescence assays were performed to determine changes in E?cadherin, vimentin and NF??B expression levels. ELISAs were performed to quantify the levels of matrix metallopeptidase (MMP)?2, MMP?9 and vascular endothelial growth factor (VEGF) in the culture medium. Western blot analysis was conducted to measure the protein expression levels of MMP?2, MMP?9, PI3K, phosphorylated (p?) PI3K, AKT, p?AKT, inhibitor of NF??? kinase (IKK) and p?IKK. The results demonstrated that the migration ability of the COX?2?overexpressing MG?63 cells was significantly increased compared with the control cells. The migration ability of cells treated with NS398 or LY294002 was significantly decreased. Compared with the control cells, E?cadherin expression was significantly decreased in COX?2?overexpressing cells, while the expression levels of vimentin, MMP?2, MMP?9, VEGF, p?PI3K, p?AKT and p?IKK were significantly increased. Compared with the control cells, E?cadherin expression was significantly increased in cells treated with NS398 or LY294002, while the expression levels of vimentin, MMP?2, MMP?9, VEGF, p?PI3K, p?AKT, and p?IKK were significantly decreased. The total protein levels of PI3K, AKT and IKK were not changed among the treatment groups. In summary, COX?2 overexpression decreased the expression levels of the epithelial protein E?cadherin and increased the expression levels of the mesenchymal proteins vimentin, MMP?2 and MMP?9, as well as promoted cell migration, by activating the PI3K/AKT/NF??B signaling pathway.

SUBMITTER: Zhang X

PROVIDER: S-EPMC6755176 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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COX‑2 promotes epithelial‑mesenchymal transition and migration in osteosarcoma MG‑63 cells via PI3K/AKT/NF‑κB signaling.

Zhang Xueliang X Qu Peng P Zhao Hui H Zhao Tong T Cao Nong N

Molecular medicine reports 20190821 4

The present study aimed to investigate the mechanism by which cyclooxygenase‑2 (COX‑2) promotes the metastasis of MG‑63 osteosarcoma cells through the PI3K/AKT/NF‑κB pathway. To achieve this, a recombinant lentivirus containing the COX‑2 gene was constructed in order to overexpress COX‑2; a recombinant lentivirus containing a control sequence was also constructed. A Transwell chamber migration assay was performed to quantify the migration of the COX‑2‑transduced cells, and of cells treated with ...[more]

PMID: 31485669

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Project description:Onosma bracteata Wall. is an important medicinal and immunity-enhancing herbs. This plant is commonly used in the preparation of traditional Ayurvedic drugs to treat numerous diseases. Inspired by the medicinal properties of this plant, the present study aimed to investigate the antiproliferative potential and the primary molecular mechanisms of the apoptotic induction against human osteosarcoma (MG-63) cells. Among all the fractions isolated from O. bracteata, ethyl acetate fraction (Obea) showed good antioxidant activity in superoxide radical scavenging assay and lipid peroxidation assay with an EC50 value of 95.12 and 80.67 µg/mL, respectively. Silica gel column chromatography of ethyl acetate (Obea) fraction of O. bracteata yielded a pure compound, which was characterized by NMR, FTIR, and HR-MS analysis and was identified as 1,2-benzene dicarboxylic acid, bis (2-methyl propyl) ester (BDCe fraction). BDCe fraction was evaluated for the antiproliferative potential against human osteosarcoma MG-63, human neuroblastoma IMR-32, and human lung carcinoma A549 cell lines by MTT assay and exhibited GI50 values of 37.53 μM, 56.05 μM, and 47.12 μM, respectively. In MG-63 cells, the BDCe fraction increased the level of ROS and simultaneously decreased the mitochondria membrane potential (MMP) potential by arresting cells at the G0/G1 phase, suggesting the initiation of apoptosis. Western blotting analysis revealed the upregulation of p53, caspase3, and caspase9 while the expressions of p-NF-κB, p-Akt and Bcl-xl were decreased. RT-qPCR studies also showed upregulation in the expression of p53 and caspase3 and downregulation in the expression of CDK2, Bcl-2 and Cyclin E genes. Molecular docking analysis displayed the interaction between BDCe fraction with p53 (-151.13 kcal/mol) and CDK1 (-133.96 kcal/mol). The results of the present work suggest that the BDCe fraction has chemopreventive properties against osteosarcoma (MG-63) cells through the induction of cell cycle arrest and apoptosis via Akt/NF-κB/p53 pathways. This study contributes to the understanding of the utilization of BDCe fraction in osteosarcoma treatment.

Project description:BackgroundPhytoestrogens such as genistein, the most prominent isoflavone from soy, show concentration-dependent anti-estrogenic or estrogenic effects. High genistein concentrations (>10 μM) also promote proliferation of bone cancer cells in vitro. On the other hand, the most active component of the vitamin D family, calcitriol, has been shown to be tumor protective in vitro and in vivo. The purpose of this study was to examine a putative synergism of genistein and calcitriol in two osteosarcoma cell lines MG-63 (early osteoblast), Saos-2 (mature osteoblast) and primary osteoblasts.MethodsThus, an initial screening based on cell cycle phase alterations, estrogen (ER) and vitamin D receptor (VDR) expression, live cell metabolic monitoring, and metabolomics were performed.ResultsExposure to the combination of 100 μM genistein and 10 nM calcitriol reduced the number of proliferative cells to control levels, increased ERß and VDR expression, and reduced extracellular acidification (40%) as well as respiratory activity (70%), primarily in MG-63 cells. In order to identify the underlying cellular mechanisms in the MG-63 cell line, metabolic profiling via GC/MS technology was conducted. Combined treatment significantly influenced lipids and amino acids preferably, whereas metabolites of the energy metabolism were not altered. The comparative analysis of the log2-ratios revealed that after combined treatment only the metabolite ethanolamine was highly up-regulated. This is the result: a strong overexpression (350%) of the enzyme sphingosine-1-phosphate lyase (SGPL1), which irreversibly degrades sphingosine-1-phosphate (S1P), thereby, generating ethanolamine. S1P production and secretion is associated with an increased capability of migration and invasion of cancer cells.ConclusionFrom these results can be concluded that the tumor promoting effect of high concentrations of genistein in immature osteosarcoma cells is reduced by the co-administration of calcitriol, primarily by the breakdown of S1P. It should be tested whether this anti-metastatic pathway can be stimulated by combined treatment also in metastatic xenograft mice models.

Dataset Information

COX?2 promotes epithelial?mesenchymal transition and migration in osteosarcoma MG?63 cells via PI3K/AKT/NF??B signaling.

Publications

COX‑2 promotes epithelial‑mesenchymal transition and migration in osteosarcoma MG‑63 cells via PI3K/AKT/NF‑κB signaling.

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