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Heme Oxygenase-1 and Carbon Monoxide Regulate Growth and Progression in Glioblastoma Cells

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Abstract

In human glioma tumours, heme oxygenase-1 (HO-1) is overexpressed when compared with normal brain tissues and during oligodendroglioma progression. However, the molecular mechanisms mediated by HO-1 to promote glioblastoma remain unknown. We therefore aimed at investigating the effect of HO-1 expression and its selective enzymatic inhibition in two different cell lines (i.e. A172 and U87-MG). HO-1 was induced by hemin treatment (10 μM), and VP13/47 (100 μM) was used as a specific non-competitive inhibitor of HO-1 activity. Cell proliferation was measured by cell index measurement (xCelligence technology) and clonogenic assay, whereas cell migration was assessed by wound healing assay. Carbon monoxide-releasing molecules (CORMs) (i.e. CORM-3 and CORM-A1) were also used in a separate set of experiments to confirm the effect of HO-1 by-product in glioblastoma progression further. Our results were further validated using GSE4412 microarray dataset analysis and comparing biopsies overexpressing HO-1 with the rest of the cases. Our results showed that hemin was able to induce both HO-1 gene and protein expression in a cell-dependent manner being A172 more responsive to pharmacological upregulation of HO-1. Hemin, but not CORMs treatment, resulted in a significant increase of cell proliferation following 24 h of treatment as measured by increased cell index and colony formation capacity and such effect was abolished by VP13/47. Interestingly, both hemin and CORMs showed a significant effect on the wound healing assay also exhibiting cell specificity. Finally, our dataset analysis showed a positive correlation of HO-1 gene expression with ITGBI and ITGBII which are membrane receptors involved in cell adhesion, embryogenesis, tissue repair, immune response and metastatic diffusion of tumour cells. In conclusion, our data suggest that HO-1 and its by-product CO exhibit a cell-specific effect on various aspects of disease progression and are associated with a complex series of molecular mechanisms driving cell proliferation, survival and metastasis.

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Funding

This work was supported by Research Funding for University of Catania, Italy (Piano per la Ricerca, FIR 2018-2020). This work was part of the PhD thesis of Dr. Carlo Castruccio Castracani (Neuroscience International PhD program).

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CCC and GLV made a substantial contribution to the concept and design, acquisition of data or analysis and interpretation of data; GLV drafted the article or revised it critically for important intellectual content; LL, AD, GL, DC, DT, VP and GLV performed in vitro experiments; MDR: analysed GEO datasets and performed statistical analysis; all the authors approved the version to be published.

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Correspondence to Giovanni Li Volti.

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Supplementary Figure 1.

Expression levels of HO-1 in several brain tumours. Gene expression profiles were used to identify HO-1 gene that is differentially expressed between various brain tumour types. Data are expressed as z-score intensity expression levels and presented as vertical scatter dot plots and violin plots. P values <0.05 were considered to be statistically significant (*p<0.05; **p<0.005; ***p<0.0005; ****p<0.00005). (PNG 534 kb)

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Castruccio Castracani, C., Longhitano, L., Distefano, A. et al. Heme Oxygenase-1 and Carbon Monoxide Regulate Growth and Progression in Glioblastoma Cells. Mol Neurobiol 57, 2436–2446 (2020). https://doi.org/10.1007/s12035-020-01869-7

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