Comparative gene expression profiling of human metallothionein-3 up-regulation in neuroblastoma cells and its impact on susceptibility to cisplatin

Abstract

Human metallothionein-3 (hMT-3), also known as growth inhibitory factor, is predominantly expressed in the central nervous system. hMT-3 is presumed to participate in the processes of heavy metal detoxification, regulation of metabolism and protection against oxidative damage of free radicals in the central nervous system; thus, it could play important neuromodulatory and neuroprotective roles. However, the primary functions of hMT-3 and the mechanism underlying its multiple functions in neuroblastoma have not been elucidated so far. First, we confirmed relatively high expression of hMT-3 encoding mRNA in biopsies (n = 23) from high-risk neuroblastoma subjects. Therefore, we focused on investigation of the impact of hMT-3 up-regulation in N-Myc amplifying neuroblastoma cells. The differentially up-regulated genes involved in biological pathways related to cellular senescence and cell cycle were identified using electrochemical microarray with consequent bioinformatic processing. Further, as experimental verification of microarray data, the cytotoxicity of the cisplatin (CDDP) was examined in hMT-3 and mock cells by MTT and clonogenic assays. Overall, our data strongly suggest that up-regulation of hMT-3 positively correlates with the genes involved in oncogene-induced senescence (CDKN2B and ANAPC5) or apoptosis (CASP4). Moreover, we identified a significant increase in chemoresistance to cisplatin (CDDP) due to hMT-3 up-regulation (24IC50: 7.5 vs. 19.8 μg/ml), indicating its multipurpose biological significance.

Transcriptomic Landscape of Cisplatin-Resistant Neuroblastoma Cells

“…We revalidated the marked phenotypical alterations in UKF-NB-4 CDDP cells compared to the parental cells [22,31]. Figure 1A illustrates that induction of CDDP chemoresistance results in pronounced morphological changes from initial polygonal morphology of UKF-NB-4 cells to more globular and less-elongated UKF-NB-4 CDDP cells.…”

“…The list of primers used for validation of microarray data is shown in Table S2. The qPCR experiments were performed in conditions described in our previous study [22].…”

“…The delineation of novel molecular targets that participate on the aggressiveness of Nbl, together with the development of agents that will influence the functionality of such targets, is of utmost interest towards improvement of patient survival [21]. Our previous study demonstrated that the transient up-regulation of metallothionein-3 (MT-3) in Nbl cells potently induces chemoresistance to CDDP, revealing a great potential for further investigation of MTs for use as biomarkers of CDDP [22]. CDDP affects several important pathways, which stimulate molecular mechanisms, which are responsible for the development of CDDP chemoresistance by developing a complex self-defense network to escape exogenous cytotoxic substances of distinct origin.…”

Towards an Understanding of Specific Response of Metallothionein (Sub)Isoforms to Exposure to Platinum-Based Anticancer Drugs

“…Merlos Rodrigo et al (2017) conclude that cisplatin inducts cytotoxic cell death through apoptotic mechanism and via mitochondrial pathways. Cisplatin interacts both with DNA and with proteins.…”

“…This mechanism results in the decrease of the drug concentration below the effective level. Thus, MTs can inhibit the platinum-based cancer drug (Merlos Rodrigo et al, 2017). MTs are suspected to interfere with the metal-based drugs through ligand removal and metal sequestration.…”