Abstract
Background: Resistance of cancer cells to cytostatics is caused by a number of mechanisms that are often combined. These include reduced cell entry or increased efflux, increased DNA repair, defects of, apoptotic pathways, increased cytostatic degradation as well as elevated levels of intracellular thiols of glutathione and metallothioneins (MT).
It has been reported that high concentrations of thiol groups in the cytoplasm bind platinum alkylation derivatives and chemorezistence is due to the transfer of platinum from the cytostatic to MT, which inactivates them. Because we have shown an increase in MT levels in resistant neuroblastoma (NB) lines, but not in sensitive lines after incubation with platinum cytostatics, we have considered MT-3 for NB cells in our previous studies.
Method: SiMa NB cell lines transfected with vector containing human MT-3 and GFP or GFP only (control). Expression Microarray Human Cancer 3711 ElectraSense medium density 4 x 2k array slides with 1,609 DNA probes (Custom Array, Bothell, WA, USA), MT-3 expression and most expressed genes validated by real-time polymerase chain reaction.
Sensitivity to CDDP (cisplatin) - MTT assay, clonogenicity test, Western blott caspase cleavage and free oxygen radicals fluorescence microscopy after CellROX Deep Red Reagent staining. Levels of MT-3 mRNA in 23 samples of high-risk NB, normal human cortex and bovine adrenal glands were investigated by reverse transcription polymerase chain reaction.
Results: Expression microarray showed downregulation 3 and overexpression of 19 genes in MT-3 transfected NB cells. Using gene ontology, over-expressed genes have been shown to drive senescence-induced oncogenes (CDKN2B and ANAPC5), and the genes of glutathione S-transferase M3, caspase 4 and DNAJB6 (chaperone neuronal proteins) were also expressed.
We have demonstrated a reduced sensitivity of MT-3 transfected cells to CDDP (24h IC50 of 7.48 +- 0.97 and 19.81 +- 1.2 μg/ml), a higher number of colonies after incubation with CDDP, reduced caspase 3 after incubation with CDDP and lower free oxygen radicals after induction of CDDP. High-grade NB cells expressed MT-3 significantly more than non-tumoral adrenal cells but failed to show a clear relationship to disease course.
Conclusion: We have demonstrated the relationship between MT-3 and senescence-induced oncogene genes and some other genes relevant to cell fate (glutathione S-transferase M3, caspase 4 and DNAJB6) and a significant proportion of MT-3 on CDDP resistance. High levels of MT-3 in high-risk NB could be one of the causes of frequent relapses in this tumor.
Key words: neuroblastoma - metallothionein 3 - chemoresistance The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.