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LOX expression and functional analysis in astrocytomas and impact of IDH1 mutation.

da Silva R, Uno M, Marie SK, Oba-Shinjo SM - PLoS ONE (2015)

Bottom Line: The results demonstrate that increased expression and activity of LOX, BMP1 and HIF1A were positively correlated with the malignant grade of astrocytomas.Glioblastoma with a mutation in IDH1 expressed lower levels of LOX in the nucleus, and IDH1-mutated cases showed lower LOX expression levels when compared to wild-type IDH1 cases.Taken together, these results corroborate the role of LOX in the migration, invasion and angiogenesis of astrocytomas.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular and Cellular Biology, Department of Neurology, Faculdade de Medicina da Universidade de São Paulo, São Paulo, 01246-903, Brazil.

ABSTRACT
Lysyl oxidase (LOX) is involved in vital biological processes such as cell motility, cell signaling and gene regulation. Deregulation of this protein can contribute to tumor formation and progression. Although it is known that LOX is involved in invasion, proliferation and tumor migration in other types of tumors, studies of LOX in astrocytomas of different grades are scarce. The purpose of our study was to characterize LOX, BMP1 and HIF1A expression by real-time PCR in astrocytomas with WHO grades I to IV compared to non-neoplastic brain tissue. IDH1 mutational status was determined by PCR and sequencing. LOX protein expression was also analyzed by immunohistochemistry. LOX functional analyses were performed using siRNA knockdown and the specific inhibitor BAPN in two glioblastoma cell lines. The expression levels of LOX, BMP1 and HIF1A were correlated and analyzed according to IDH1 mutation status and to the clinical end-point of overall survival of glioblastoma patients. The results demonstrate that increased expression and activity of LOX, BMP1 and HIF1A were positively correlated with the malignant grade of astrocytomas. LOX protein expression also increased according to the degree of malignancy, with localization in the cytoplasm and nucleus and staining observed in endothelial cells. Glioblastoma with a mutation in IDH1 expressed lower levels of LOX in the nucleus, and IDH1-mutated cases showed lower LOX expression levels when compared to wild-type IDH1 cases. LOX knockdown and inhibition by BAPN in U87MG and A172 cell lines affected migration, invasion and soft agar colony formation. Taken together, these results corroborate the role of LOX in the migration, invasion and angiogenesis of astrocytomas. Furthermore, LOX expression is influenced by IDH1 mutational status. This work provides new insights for researchers aiming to design targeted therapies to control astrocytomas.

No MeSH data available.


Related in: MedlinePlus

Effect of LOX knockdown on the invasive phenotype and anchorage-independent growth behavior of GBM cell lines.Evaluation of the invasion (A and B) and anchorage-independent growth (C and D) of U87MG and A172 cell lines after LOX silencing with siRNA were compared to the non-targeted control (NTC). A total of 2.5 x 104 cells were seeded in the upper chamber of Transwell inserts. Cells in the lower chamber were fixed and stained with crystal violet after 18 hours of incubation. Graphs represent the number of migrated cells per field in each condition (means ± standard errors of the means) in the two independent experiments conducted in duplicate. The images show representative fields of U87MG (A) and A172 (B) cells that invaded and crossed the insert (40x magnification). Soft agar colony formation assays were performed for both U87MG (C) and A172 (D) cell lines with 1 x 103 cells. Graphs represent the mean number of colonies ± SD of two independent experiments conducted in duplicate (Mann-Whitney test, **p<0.001; ***p<0.0001).
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pone.0119781.g004: Effect of LOX knockdown on the invasive phenotype and anchorage-independent growth behavior of GBM cell lines.Evaluation of the invasion (A and B) and anchorage-independent growth (C and D) of U87MG and A172 cell lines after LOX silencing with siRNA were compared to the non-targeted control (NTC). A total of 2.5 x 104 cells were seeded in the upper chamber of Transwell inserts. Cells in the lower chamber were fixed and stained with crystal violet after 18 hours of incubation. Graphs represent the number of migrated cells per field in each condition (means ± standard errors of the means) in the two independent experiments conducted in duplicate. The images show representative fields of U87MG (A) and A172 (B) cells that invaded and crossed the insert (40x magnification). Soft agar colony formation assays were performed for both U87MG (C) and A172 (D) cell lines with 1 x 103 cells. Graphs represent the mean number of colonies ± SD of two independent experiments conducted in duplicate (Mann-Whitney test, **p<0.001; ***p<0.0001).

Mentions: Transfected cells were maintained under cell culture conditions for 2, 4 and 7 days after transfection to evaluate the involvement of LOX in the proliferation of A172 and U87MG cells. There was no difference in the proliferation rate of cells after LOX knockdown when compared to NTC in both cell lines analyzed (data not shown). To test the hypothesis that LOX expression was correlated with the migratory ability of tumor cells, U87MG and A172 cell lines were evaluated after knocking down LOX expression by siRNA and after inhibiting the active form of LOX with a specific drug (BAPN). There was a reduction in the migration ability of U87MG and A172 cells after either transfection with siRNA for LOX or treatment with BAPN, as shown in Fig. 3C and 3D. The differences between the LOX siRNA and NTC groups were statistically significant in both U87MG (p<0.001) and A172 (p<0.0001) cells. Inhibition of LOX by BAPN significantly inhibited the migration of both U87MG and A172 cells when compared to non-treated cells (p<0.0001 for both cell lines). LOX involvement in the invasion of GBM cell lines was also observed for U87MG and A172 cells (Fig. 4A and 4B) (p<0.0001 for both).


LOX expression and functional analysis in astrocytomas and impact of IDH1 mutation.

da Silva R, Uno M, Marie SK, Oba-Shinjo SM - PLoS ONE (2015)

Effect of LOX knockdown on the invasive phenotype and anchorage-independent growth behavior of GBM cell lines.Evaluation of the invasion (A and B) and anchorage-independent growth (C and D) of U87MG and A172 cell lines after LOX silencing with siRNA were compared to the non-targeted control (NTC). A total of 2.5 x 104 cells were seeded in the upper chamber of Transwell inserts. Cells in the lower chamber were fixed and stained with crystal violet after 18 hours of incubation. Graphs represent the number of migrated cells per field in each condition (means ± standard errors of the means) in the two independent experiments conducted in duplicate. The images show representative fields of U87MG (A) and A172 (B) cells that invaded and crossed the insert (40x magnification). Soft agar colony formation assays were performed for both U87MG (C) and A172 (D) cell lines with 1 x 103 cells. Graphs represent the mean number of colonies ± SD of two independent experiments conducted in duplicate (Mann-Whitney test, **p<0.001; ***p<0.0001).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4366168&req=5

pone.0119781.g004: Effect of LOX knockdown on the invasive phenotype and anchorage-independent growth behavior of GBM cell lines.Evaluation of the invasion (A and B) and anchorage-independent growth (C and D) of U87MG and A172 cell lines after LOX silencing with siRNA were compared to the non-targeted control (NTC). A total of 2.5 x 104 cells were seeded in the upper chamber of Transwell inserts. Cells in the lower chamber were fixed and stained with crystal violet after 18 hours of incubation. Graphs represent the number of migrated cells per field in each condition (means ± standard errors of the means) in the two independent experiments conducted in duplicate. The images show representative fields of U87MG (A) and A172 (B) cells that invaded and crossed the insert (40x magnification). Soft agar colony formation assays were performed for both U87MG (C) and A172 (D) cell lines with 1 x 103 cells. Graphs represent the mean number of colonies ± SD of two independent experiments conducted in duplicate (Mann-Whitney test, **p<0.001; ***p<0.0001).
Mentions: Transfected cells were maintained under cell culture conditions for 2, 4 and 7 days after transfection to evaluate the involvement of LOX in the proliferation of A172 and U87MG cells. There was no difference in the proliferation rate of cells after LOX knockdown when compared to NTC in both cell lines analyzed (data not shown). To test the hypothesis that LOX expression was correlated with the migratory ability of tumor cells, U87MG and A172 cell lines were evaluated after knocking down LOX expression by siRNA and after inhibiting the active form of LOX with a specific drug (BAPN). There was a reduction in the migration ability of U87MG and A172 cells after either transfection with siRNA for LOX or treatment with BAPN, as shown in Fig. 3C and 3D. The differences between the LOX siRNA and NTC groups were statistically significant in both U87MG (p<0.001) and A172 (p<0.0001) cells. Inhibition of LOX by BAPN significantly inhibited the migration of both U87MG and A172 cells when compared to non-treated cells (p<0.0001 for both cell lines). LOX involvement in the invasion of GBM cell lines was also observed for U87MG and A172 cells (Fig. 4A and 4B) (p<0.0001 for both).

Bottom Line: The results demonstrate that increased expression and activity of LOX, BMP1 and HIF1A were positively correlated with the malignant grade of astrocytomas.Glioblastoma with a mutation in IDH1 expressed lower levels of LOX in the nucleus, and IDH1-mutated cases showed lower LOX expression levels when compared to wild-type IDH1 cases.Taken together, these results corroborate the role of LOX in the migration, invasion and angiogenesis of astrocytomas.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular and Cellular Biology, Department of Neurology, Faculdade de Medicina da Universidade de São Paulo, São Paulo, 01246-903, Brazil.

ABSTRACT
Lysyl oxidase (LOX) is involved in vital biological processes such as cell motility, cell signaling and gene regulation. Deregulation of this protein can contribute to tumor formation and progression. Although it is known that LOX is involved in invasion, proliferation and tumor migration in other types of tumors, studies of LOX in astrocytomas of different grades are scarce. The purpose of our study was to characterize LOX, BMP1 and HIF1A expression by real-time PCR in astrocytomas with WHO grades I to IV compared to non-neoplastic brain tissue. IDH1 mutational status was determined by PCR and sequencing. LOX protein expression was also analyzed by immunohistochemistry. LOX functional analyses were performed using siRNA knockdown and the specific inhibitor BAPN in two glioblastoma cell lines. The expression levels of LOX, BMP1 and HIF1A were correlated and analyzed according to IDH1 mutation status and to the clinical end-point of overall survival of glioblastoma patients. The results demonstrate that increased expression and activity of LOX, BMP1 and HIF1A were positively correlated with the malignant grade of astrocytomas. LOX protein expression also increased according to the degree of malignancy, with localization in the cytoplasm and nucleus and staining observed in endothelial cells. Glioblastoma with a mutation in IDH1 expressed lower levels of LOX in the nucleus, and IDH1-mutated cases showed lower LOX expression levels when compared to wild-type IDH1 cases. LOX knockdown and inhibition by BAPN in U87MG and A172 cell lines affected migration, invasion and soft agar colony formation. Taken together, these results corroborate the role of LOX in the migration, invasion and angiogenesis of astrocytomas. Furthermore, LOX expression is influenced by IDH1 mutational status. This work provides new insights for researchers aiming to design targeted therapies to control astrocytomas.

No MeSH data available.


Related in: MedlinePlus