Using Genomic And Proteomic Techniques To Study The Protective Effect Of Metallothionein Against Doxorubicin-induced Cardiotoxicity

Doxorubicin (DOX), an anthracycline antibiotic, is one of the most effective and important anticancer agents widely used in the treatment of a variety of human cancers including acute leukaemia and malignant lymphoma as well as a number of solid tumors. However, the clinical application of DOX was limited by its dose-dependent side effect of cardiotoxicity. Thus, it is important to investigate the molecular mechanism of DOX cardiotoxicity and find out the effective measures to reduce the cardiotoxicity of DOX. Metallothionein (MT) is a low molecular weight protein characterized by its high content of cysteine residues. MT exists in most tissues of a wide variety of vertebrate and invertebrate species and involved in various physiological and pathological processes. It has been reported that MT is an endogenous protective agent against many factors-induced oxidative injuries including ionizing radiation, inflammation, infection and electrophillic anti cancer drugs and so on. However the precise mechanism involved in the protective effect of MT remain an enigma. In mammals, MT binds predominantly zinc. While under the conditions of copper or calcium overload, zinc can be readily displaced by these metals. The expression of MT can be highly induced by an array of factors including heavy metals, cytokines, shock, oxidative stress agents, and among which zinc is a potent and safe inductor of MT. Recent years MT-null mice provide a useful experimental model for the deep investigation of biological function of MT. Therefore this study used MT-null (MT-/-) mice and the corresponding wild type mice (MT+/+) pretreated with or without zinc to establish the mouse model with different expression level of MT to investigate the protective effect of MT against DOX-induced cardiac injuries as well as the involved possible mechanisms. In addition, combined with the traditional technology of toxicological study, proteomics and genomics techniques were also applied in this study to analyze the global changes of genes and proteins, thus to explore the possible key genes and proteins as well as pathways responsible for the protective effect of MT against DOX-induced cardiotoxicity.Both MT+/+ and MT-/- mice were pretreated with either saline or zinc (300μmol/kg, s.c., once a day for 2 days) prior to a single dose of DOX (15 mg/kg, i.p.) or equal volume of saline. Animals were sacrificed on the 4th day after DOX administration and samples were collected for further analyses. DOX caused remarkable cardiac damage in both MT+/+ and MT-/- mice as demonstrated by the increase of serum biochemical parameter (CK, LDH and cTnT) and cardiac MDA content as well as the histophathological alterations. Zinc pretreatment significantly increased the cardiac MT levels and therefore inhibited the cardiac toxic effects of DOX only in MT+/+ mice, but not in MT-/- mice. Furthermore, Elevated formation of superoxide and peroxynitrite were obviously observed after DOX treatment, while these elevation were prevented by MT induction by zinc in MT+/+ mice, but not in MT-/- mice. These findings suggest that metallothionein induction by zinc exhibits protective effects on the cardiac toxicology of DOX, which might be mediated through the prevention of superoxide generation and related nitrosative impairment.Global transcripts analysis by GeneChip Mouse Genome 430 2.0 revealed that,①Under physiological condition, there were 10 differentially expressing genes (DE genes) between the hearts of MT-/- mice and MT+/+ mice, and these 10 DE genes were involved in cytokine-cytokine receptor interaction and Jak-STAT signaling pathway.②After DOX administration, there were 50 DE genes between the hearts of MT-/- mice and MT+/+ mice, among which 8 DE genes coded membrane components, 7 DE genes coded mitochondrial components, 3 DE genes coded endoplasmic reticulum components. Moreover, the representative DE genes among these 50 DE genes included ucp3, calr3, ugt 1 family, prx2 and so on. (3) After the comparison of MT+/+ mice with DOX vs MT +/+ control mice, and MT-/- mice with DOX vs MT -/- control mice, further analysis of the DE genes obtained from these two above comparisons revealed that there were 483 genes with different trend of change. For example 32 DE genes upregulated in the comparison of MT+/+ mice with DOX vs MT +/+ control mice, while them did not change in the comparison of MT-/- mice with DOX vs MT -/- control mice. These 483 DE genes involved in pathways related to amino acid metabolism, nucleotide metabolism, carbohydrate metabolism, oxidative phosphorylation, glutathione metabolism and so on. These findings suggest that under physiological condition the responsive potential were different between MT+/+ mice and MT-/- mice, which leading to the different results upon DOX administration. Added to this, the above mentioned genes and pathways might be the key points during MT protective effect against DOX-induced cardiotoxicity.An expression proteomic analysis involving two-dimensional electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass Spectrometry was used to identify MT-induced changes in cytoprotection-related proteins. 13 proteins were identified , which namely Acyl-Coenzyme A dehydrogenase short chain, Isovaleryl coenzyme A dehydrogenase, Peroxiredoxin 3&6, Prohibitin, crystallin alpha B, Acetyl-Coenzyme A dehydrogenase, medium chain, ATP synthase, Ubiquinol-cytochrome c reductase core protein 1, Myosin light polypeptide 4, Triosephosphate isomerase 1, ES1 protein and Enolase 3 beta muscle. Among which the results of Peroxiredoxin family and Prohibitin obtained from proteomic analysis were in accord with the results of gene transcription level obtained from genomic analysis.