RESUMO
BACKGROUND: NME6 is a member of the nucleoside diphosphate kinase (NDPK/NME/Nm23) family which has key roles in nucleotide homeostasis, signal transduction, membrane remodeling and metastasis suppression. The well-studied NME1-NME4 proteins are hexameric and catalyze, via a phospho-histidine intermediate, the transfer of the terminal phosphate from (d)NTPs to (d)NDPs (NDP kinase) or proteins (protein histidine kinase). For the NME6, a gene/protein that emerged early in eukaryotic evolution, only scarce and partially inconsistent data are available. Here we aim to clarify and extend our knowledge on the human NME6. RESULTS: We show that NME6 is mostly expressed as a 186 amino acid protein, but that a second albeit much less abundant isoform exists. The recombinant NME6 remains monomeric, and does not assemble into homo-oligomers or hetero-oligomers with NME1-NME4. Consequently, NME6 is unable to catalyze phosphotransfer: it does not generate the phospho-histidine intermediate, and no NDPK activity can be detected. In cells, we could resolve and extend existing contradictory reports by localizing NME6 within mitochondria, largely associated with the mitochondrial inner membrane and matrix space. Overexpressing NME6 reduces ADP-stimulated mitochondrial respiration and complex III abundance, thus linking NME6 to dysfunctional oxidative phosphorylation. However, it did not alter mitochondrial membrane potential, mass, or network characteristics. Our screen for NME6 protein partners revealed its association with NME4 and OPA1, but a direct interaction was observed only with RCC1L, a protein involved in mitochondrial ribosome assembly and mitochondrial translation, and identified as essential for oxidative phosphorylation. CONCLUSIONS: NME6, RCC1L and mitoribosomes localize together at the inner membrane/matrix space where NME6, in concert with RCC1L, may be involved in regulation of the mitochondrial translation of essential oxidative phosphorylation subunits. Our findings suggest new functions for NME6, independent of the classical phosphotransfer activity associated with NME proteins.
RESUMO
Glioblastoma (GBM) is the most common type of primary tumor of the central nervous system with a poor prognosis, needing the development of new therapeutic drugs. Few studies focused on sodium selenite (SS) effects in cancer cells cultured as multicellular tumor spheroids (MCTS or 3D) closer to in vivo tumor. We investigated SS anticancer effects in three human GBM cell lines cultured in 3D: LN229, U87 (O(6)-methyguanine-DNA-methyltransferase (MGMT) negative) and T98G (MGMT positive). SS absorption was evaluated and the cytotoxicity of SS and temozolomide (TMZ), the standard drug used against GBM, were compared. SS impacts on proliferation, cell death, and invasiveness were evaluated as well as epigenetic modifications by focusing on histone deacetylase (HDAC) activity and dimethyl-histone-3-lysine-9 methylation (H3K9m2), after 24h to 72h SS exposition. SS was absorbed by spheroids and was more cytotoxic than TMZ (i.e., for LN229, the IC50 was 38 fold-more elevated for TMZ than SS, at 72h). SS induced a cell cycle arrest in the S phase and apoptosis via caspase-3. SS decreased carbonic anhydrase-9 (CA9) expression, invasion on a Matrigel matrix and modulated E- and N-Cadherin transcript expressions. SS decreased HDAC activity and modulated H3K9m2 levels. 3D model provides a relevant strategy to screen new drugs and SS is a promising drug against GBM that should now be tested in GBM animal models.