The effects of extracellular matrix glycosylation inhibition on osteosarcoma invasiveness / Sarmila Hanim Mustafa

• Main Author: Mustafa, Sarmila Hanim
• Format: Thesis
• Language: English
• Published: 2020
• Subjects: Research. Experimentation
• Online Access: https://ir.uitm.edu.my/id/eprint/60051/1/60051.pdf; https://ir.uitm.edu.my/id/eprint/60051/

Osteosarcoma (OS) is a rare malignant bone cancer affecting children and young adults. To date, OS still has a poor prognosis despite the use of advanced multimodal therapy in its treatment. Recurrence cases has also shown to be increased in metastatic patients especially to the lung. A possible cause for this poor prognosis could be because of lack of understanding in the underlying mechanism of OS progression and metastasis. Previously, loss of Wnt/β-catenin pathway activity during osteoblast differentiation was suggested to contribute to the OS development. In addition, a metastatic condition of OS was shown to be established through the adhesion of OS cells to blood vessel mediated by α4β1 integrin. It has been known that aberrant glycosylation is associated with metastasis in various cancer by modulating the cells invasiveness and their dissemination through the extracellular matrix (ECM) layer. However, the effect of aberrant glycosylation, mainly during N-glycan processing in OS has yet to be elucidated. Therefore, the objective of this study is to investigate the effects of aberrant glycosylation towards OS invasiveness via the cell-ECM interaction. In this study, the inhibition of glycosylation was carried out by the treatment of OS cell line (MG-63) with 1-deoxynojirimycin (1-DNJ); an α- glucosidase-I/II inhibitor, and 1-deoxymannojirimycin (1-DMJ); an α-mannosidase I inhibitor. Then, its effects on glycosylation pattern, invasion ability, protein expression of α4β1 integrin and the expression level of ECM degradative enzymes genes; MMP-2, MMP-9, TIMP-1, TIMP-2 and β-catenin genes were determined. In this study, the normal osteoblast cell (hFOB1.19) was also treated with 1-DNJ and 1- DMJ and both samples were subjected to the same method as MG-63 cells except for cell invasion assay. The results showed that the inhibition of N-glycosylation by 1- DMJ enhanced the invasion of MG-63 cells through the ECM layer significantly by 3.87 fold (p<0.05). It was also found that 1-DMJ significantly (p<0.05) regulates the expression of α4β1 integrin protein in hFOB1.19 cells. qRT-PCR analysis showed that 1-DMJ downregulate the expression MMP-9 gene (2.42 fold ± 0.284; p<0.05) in MG- 63 cells. On the other hand, TIMP-1 (1.54 fold ± 0.035), TIMP-2 (1.22 fold ± 0.041) and β-catenin (1.24 fold ± 0.07) genes were significantly (p<0.05) upregulated in MG- 63 cells when compared to control. However, in hFOB1.19 cells treated with 1-DMJ, MMP-2 (48.32 fold ± 0.16), MMP-9 (4.38 fold ± 0.389), and TIMP-1 (1.45 fold ± 0.064) genes were significantly (p<0.05) downregulated when compared to control. Whereas, the expression of TIMP-2 (1.2 fold ± 0.03), and β-catenin gene (2.22 fold ± 0.232) in hFOB1.19 cells were significantly (p<0.05) upregulated. 1-DNJ was shown to downregulate the expression of MMP-2 in both cells; MG-63 (1.29 fold ± 0.067) and hFOB1.19 (55.25 fold ± 0.097) significantly (p<0.05) when compared to control. However, the expression of β-catenin gene in 1-DNJ treated hFOB1.19 cells was significantly (p<0.05) increased by 2.4 fold ± 0.103 when compared to control. This study suggested that 1-DMJ may promote the invasion of MG-63 cells through the ECM layer via the regulation of MMP-9, TIMP-1, and TIMP-2 genes. Whereas the overexpression of β-catenin genes in 1-DMJ and 1-DNJ treated hFOB1.19 cells might give an insight on the epithelial-to-mesenchymal transition (EMT) of normal osteoblast cells. This study might provide more information on the underlying mechanism of OS therefore contributes to OS prognosis improvement.