Clinical significance of metallothioneins in cell therapy and nanomedicine

Abstract

Mammalian metallothioneins (MTs) are low molecular weight (6-7 kDa) cysteine-rich proteins that are specifically induced by metal nanoparticles (NPs). MT induction in cell therapy may provide better protection by serving as antioxidant, anti-inflammatory, antiapoptotic agents, and by augmenting zinc-mediated transcriptional regulation of genes involved in cell proliferation and differentiation. Liposome-encapsulated MT-1 promoter has been used extensively to induce growth hormone or other genes in culture and gene-manipulated animals. MTs are induced as a defensive mechanism in chronic inflammatory conditions including neurodegenerative diseases, cardiovascular diseases, cancer, and infections, hence can serve as early and sensitive biomarkers of environmental safety and effectiveness of newly developed NPs for clinical applications. Microarray analysis has indicated that MTs are significantly induced in drug resistant cancers and during radiation treatment. Nutritional stress and environmental toxins (eg, kainic acid and domoic acid) induce MTs and aggregation of multilamellar electron-dense membrane stacks (Charnoly body) due to mitochondrial degeneration. MTs enhance mitochondrial bioenergetics of reduced nicotinamide adenine dinucleotide-ubiquinone oxidoreductase (complex-1), a rate-limiting enzyme complex involved in the oxidative phosphorylation. Monoamine oxidase-B inhibitors (eg, selegiline) inhibit α-synuclein nitration, implicated in Lewy body formation, and inhibit 1-methyl 4-phenylpyridinium and 3-morpholinosydnonimine-induced apoptosis in cultured human dopaminergic neurons and mesencephalic fetal stem cells. MTs as free radical scavengers inhibit Charnoly body formation and neurodegenerative α-synucleinopathies, hence Charnoly body formation and α-synuclein index may be used as early and sensitive biomarkers to assess NP effectiveness and toxicity to discover better drug delivery and surgical interventions. Furthermore, pharmacological interventions augmenting MTs may facilitate the theranostic potential of NP-labeled cells and other therapeutic agents. These unique characteristics of MTs might be helpful in the synthesis, characterization, and functionalization of emerging NPs for theranostic applications. This report highlights the clinical significance of MTs and their versatility as early, sensitive biomarkers in cell-based therapy and nanomedicine.

Keywords: Charnoly body; free radicals; metallothioneins; nanomedicine; stem cells; theranostics; toxicity; α-synuclein index.

Figure 1

(A) Transmission electron microscope (magnification 50,000×) image illustrating CB formation in the developing undernourished Purkinje neurons as a consequence of degeneration of mitochondrial membranes (a). These membranes are transformed in to multilamellar (usually pentalamellar or heptalamellar) electron-dense structures (b) to form mature CBs (c). The structural degradation of CBs occurs eventually due to increased lysosomal activity (d). Based on the transmission electron microscopy and electrophysiological studies, it is proposed that CBs are involved in desmosomal repair, autophagy, and increased afterhyperpolarization duration due to increased intracellular calcium ions (Ca²⁺) in the 15-day undernourished rat cerebellar Purkinje neurons. (B) Free radicals (indicated by dots) are generated as a byproduct of mitochondrial oxidative phosphorylation. Notes: Toxic NPs, environmental toxins, chemicals, drugs, nutritional stress, and infections augment free radical overproduction, which may trigger CB formation. MTs serve as free radical scavengers to protect mitochondrial structural and functional integrity by inhibiting CB formation. CB formation is a transitory state between apoptosis and cell death and is a reversible process. (+) indicates activation; (−) indicates inhibition. Abbreviations: CB, Charnoly body; MT, metallothionein; NP, nanoparticle.

Figure 2

Three types of NPs have been proposed (1) toxic, (2) protective, and (3) inert. NPs augmenting the zinc ion homeostasis could be neuroprotective, those inducing dyshomeostasis could be toxic, and those producing no significant change could be neutral or inert. Thus, metal ion speciation of MTs following NP exposure employing sensitive procedure such as inductively coupled plasma mass spectroscopy may provide a better understanding of the therapeutic potential of MTs in nanomedicine. At the ultrastructural level, protective NPs may prevent CB formation and toxic NPs will induce CB formation, whereas inert NPs will remain ineffective. Stem cells provide MT-mediated mitochondrial protection and inhibit CB formation to facilitate regenerative process. Any physical, physiological, and/or pharmacological intervention involving MT induction will inhibit CB formation and provide neuroprotection. Thus, MT induction and CB formation may be utilized as early and sensitive biomarkers of NPs safety, effectiveness, and toxicity for theranostic applications. (The specificity of these biomarkers may be explored in the future). Furthermore, toxic NPs and environmental stress may induce overproduction of free radicals (OH, NO) to cause lysosomal membrane destabilization and CB formation. Free radicals may induce lipases, proteases, and nucleases to cause membranolysis, proteolysis, and nuclear DNA depolymerization leading to apoptosis, necrosis, and even cell death. MTs may serve as free radical scavengers to inhibit CB formation and provide therapeutic effect in neurodegenerative disease and other disorders. (A) Transmission electron microscope picture (magnification 20,000×) demonstrating the cytoplasmic organization of Purkinje neuron from the undernourished rat cerebellar cortex demonstrating increased incidence of lysosomes (▼). (B) Multilamellar electron-dense membrane stacks (CB ■) in the 15-day undernourished rat Purkinje neurons (magnification 50,000×). Notes: (+) indicates activation; (−) indicates inhibition. Abbreviations: CB, Charnoly body; MT, metallothionein; NO, nitric oxide; NP, nanoparticle; OH, hydroxyl; ONOO, peroxynitrite; SI, α-synuclein index.