Chitosan oligosaccharide suppresses osteosarcoma malignancy by inhibiting CEMIP via the PI3K/AKT/mTOR pathway

Abstract

Osteosarcoma is a malignant bone tumor that is prone to metastasize early and primarily affects children and adolescents. Cell migration-inducing protein (CEMIP) plays a crucial role in the progression and malignancy of various tumor diseases, including osteosarcoma. Chitosan oligosaccharide (COS), an oligomer isolated from chitin, has been found to have significant anti-tumor activity in various cancers. This study investigates the effects of COS on CEMIP expression in osteosarcoma and explores the underlying mechanism. In present study, in vitro experiments were conducted to confirm the inhibitory activity of COS on human osteosarcoma cells. Our results demonstrate that COS possesses inhibitory effects against human osteosarcoma cells and significantly suppresses CEMIP expression in vitro. Next, we studied the inhibition of the expression of CEMIP by COS and then performed bioinformatics analysis to explore the potential inhibitory mechanism of COS against signaling pathways involved in regulating CEMIP expression. Bioinformatics analysis predicted a close association between the PI3K signaling pathway and CEMIP expression and that the inhibitory effect of COS on CEMIP expression may be related to PI3K signaling pathway regulation. The results of this study show that COS treatment significantly inhibits CEMIP expression and the PI3K/AKT/mTOR signaling pathway, as observed both in vitro and in vivo. This study demonstrates that COS could inhibit the expression of CEMIP, which is closely related to osteosarcoma malignancy. This inhibitory effect may be attributed to the inhibition of the PI3K/AKT/mTOR signaling pathway in vitro and in vivo.

Keywords: CEMIP; Chitosan oligosaccharide; Osteosarcoma; PI3K; Pathway.

Fig. 1

COS inhibited the proliferation of osteosarcoma cellsn A Human osteosarcoma cells (MG63 and U2OS) were treated with different concentrations of COS for 24 h, 48 h, and 72 h, respectively, and IC50 was calculated using Graph Pad Prism 8.0. B The colony formation of MG63 and U2OS cells was evaluated after COS treatment (0, 7, 14 and, 21 mg/mL). C Flow cytometry assay was performed to analyze apoptosis rates induced by COS treatment. Apoptosis rates in COS treatment groups were significantly higher than that in the NC group. D Western blot was conducted to evaluate pro-apoptotic proteins (BAX and C-Cas3) and anti-apoptotic protein (Bcl-2). All data are represented as mean ± SD; *p < 0.05, **p < 0.01, and ***p < 0.001. The experiments were repeated 3 times independently. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Bcl-2, B-cell lymphoma-2; BAX, BCL2-Associated X; C-CAS3, cleaved caspase 3; NC, negative control; COS, Chitosan oligosaccharide

Fig. 2

COS suppressed the migration and invasion of osteosarcoma cells. A Human osteosarcoma cells (MG63 and U2OS) were treated with various concentrations (0, 7, 14, and 21 mg/mL) of COS for 24 h, and migration ability was evaluated using wound healing assay. B Transwell cell invasion assay was performed after treating osteosarcoma cells with COS for 24 h. All data are represented as mean ± SD; *p < 0.05, **p < 0.01, and ***p < 0.001. The experiments were repeated 3 times independently. Abbreviations: NC, negative control; COS, Chitosan oligosaccharide

Fig. 3

COS inhibited the expression of CEMIP in osteosarcoma cells. A MG63 and U2OS cells were treated with various doses (0, 7, 14 and, 21 mg/mL) of COS for 48 h, and mRNA expression level of CEMIP was evaluated with qRT-PCR. B The expression level of CEMIP protein of osteosarcoma cells was evaluated using Western blot followed by COS treatment. All data is expressed as mean ± SD; *p < 0.05, **p < 0.01, and ***p < 0.001. The experiments were repeated 3 times independently. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; CEMIP, Cell migration inducing protein; NC, negative control; COS, Chitosan oligosaccharide

Fig. 4

Bioinformatics prediction for possible target signaling pathways of COS in osteosarcoma. A 2D and 3D structure of COS. B A PPI network for possible target genes of COS was constructed to obtain possible edges by using STRING database. C 104 possible target genes were screened using Cytoscape software. D–F The intersection between COS target genes and human osteosarcoma-related genes was analyzed. G The primary associated pathways were screened from a key network of COS by KEGG pathway analysis. Abbreviations: PPI, protein–protein Interaction; KEGG, Kyoto Encyclopedia of Genes and Genomes; COS, Chitosan oligosaccharide

Fig. 5

Possible target diseases, biological processes, and signaling pathways of COS. Diseases, biological processes, and signaling pathways related to COS target genes were screened using KEGG database analysis. Abbreviations: KEGG, Kyoto Encyclopedia of Genes and Genomes; COS, Chitosan oligosaccharide

Fig. 6

Docking of COS. The main target proteins (AKT1, EGFR, HRAS, HSP90AA1, IGFR1, and PTK2) were docked with COS. Abbreviations: AKT1, RAC-alpha serine/threonine-protein kinase; EGFR, Epidermal growth factor receptor; HRAS, GTPase HRas; HSP90AA1, Heat shock protein HSP 90-alpha; IGFR1, Insulin-like growth factor 1 receptor; PTK2, Protein-tyrosine kinase 2; COS, Chitosan oligosaccharide

Fig. 7

CEMIP expression was inhibited by COS via suppression of the PI3K/AKT/mTOR pathway in osteosarcoma cells. Western blot A and relevant quantitative analysis B of p-PI3K, p-AKT, and p-mTOR were performed after treating osteosarcoma cells with COS (0, 7, 14, and 21 mg/mL) for 48 h. All data are represented as mean ± SD; *p < 0.05, **p < 0.01, **p < 0.001, and ****p < 0.0001. The experiments were repeated 3 times independently. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; p-PI3K, phosphorylated phosphatidylinositol 3-kinase; p-AKT, phosphorylated RAC-alpha serine/threonine-protein kinase; p-mTOR, phosphorylated mammalian target of rapamycin; NC, negative control; COS, Chitosan oligosaccharide

Fig. 8

COS inhibited the progression of osteosarcoma and the expression of CEMIP in vivo. A–E Tumor size, volume, weight, body weight and ratio of tumor weight to body weight of mice were measured respectively. F Immunohistochemistry assay was performed to evaluate the expression of CEMIP and related pathway proteins in tumor tissue. All data are represented as mean ± SD; *p < 0.05, **p < 0.01, and ***p < 0.001. The experiments were repeated 3 times independently. Abbreviations: p-PI3K, phosphorylated phosphatidylinositol 3-kinase; p-AKT, phosphorylated RAC-alpha serine/threonine-protein kinase; p-mTOR, phosphorylated mammalian target of rapamycin; CEMIP, Cell migration inducing protein; NC, negative control; COS, Chitosan oligosaccharide