Computational studies, design and synthesis of Pd(II)-based complexes: Allosteric inhibitors of the Human Topoisomerase-IIα.

Herein, a robust docking protocol was developed by using a low-cost workflow to highlight the modulation at ATPase domain from Human Topoisomerase-IIα (TOP2A) towards four novel Pd(II)-complexes bearing N,S-donor ligands. In vitro TOP2A inhibition assay confirmed the ability of them to prevent the enzyme functions into concentration ranging at 6.25-25µM. These results exhibited more effectivity than anticancer agent etoposide (35µM) and merbarone (40-50µM). The compounds were screened via Resazurin assay against MCF-7, MDA-MB-231 (Human breast), DU-145 (Human prostate), A549 (Human lung) and Cal27 (Human tongue) tumor cell lines revealing great cytotoxic effects, primarily to MCF-7 (IC50=1.81-4.46µM). As well, 1-4 exhibited their selectivity index (SI) higher than cisplatin against HEK-293 (human kidney) normal cells, at least 11.6-fold (SI1-4=1.4-5.0; SIcis=0.12). Further, Red Blood Cell hemolytic test suggested in vitro non-toxic character for compound 4, previously evaluated as the most effective TOP2A inhibitor.

SET overexpression in HEK293 cells regulates mitochondrial uncoupling proteins levels within a mitochondrial fission/reduced autophagic flux scenario.

We hypothesized that SET, a protein accumulated in some cancer types and Alzheimer disease, is involved in cell death through mitochondrial mechanisms. We addressed the mRNA and protein levels of the mitochondrial uncoupling proteins UCP1, UCP2 and UCP3 (S and L isoforms) by quantitative real-time PCR and immunofluorescence as well as other mitochondrial involvements, in HEK293 cells overexpressing the SET protein (HEK293/SET), either in the presence or absence of oxidative stress induced by the pro-oxidant t-butyl hydroperoxide (t-BHP). SET overexpression in HEK293 cells decreased UCP1 and increased UCP2 and UCP3 (S/L) mRNA and protein levels, whilst also preventing lipid peroxidation and decreasing the content of cellular ATP. SET overexpression also (i) decreased the area of mitochondria and increased the number of organelles and lysosomes, (ii) increased mitochondrial fission, as demonstrated by increased FIS1 mRNA and FIS-1 protein levels, an apparent accumulation of DRP-1 protein, and an increase in the VDAC protein level, and (iii) reduced autophagic flux, as demonstrated by a decrease in LC3B lipidation (LC3B-II) in the presence of chloroquine. Therefore, SET overexpression in HEK293 cells promotes mitochondrial fission and reduces autophagic flux in apparent association with up-regulation of UCP2 and UCP3; this implies a potential involvement in cellular processes that are deregulated such as in Alzheimer's disease and cancer.

SET overexpression decreases cell detoxification efficiency: ALDH2 and GSTP1 are downregulated, DDR is impaired and DNA damage accumulates.

Alcohol and tobacco consumption are risk factors for head and neck squamous cell carcinoma (HNSCC). Aldehyde dehydrogenase 2 (ALDH2) and glutathione S-transferase pi 1 (GSTP1) are important enzymes for cellular detoxification and low efficiencies are implicated in cancer. We assessed the potential role of SET protein overexpression, a histone acetylation modulator accumulated in HNSCC, in gene regulation and protein activity of ALDH2 and GSTP1. SET was knocked down in HN13, HN12 and Cal27, and overexpressed in HEK293 cells; ethanol and cisplatin were the chemical agents. Cells with SET overexpression (HEK293/SET, HN13 and HN12) showed lower ALDH2 and GSTP1 mRNA levels and trichostatin A increased them (real-time PCR). Ethanol upregulated GSTP1 and ALDH2 mRNAs, whereas cisplatin upregulated GSTP1 in HEK293 cells. SET-chromatin binding revealed SET interaction with ALDH2 and GSTP1 promoters, specifically via SET NAP domain; ethanol and cisplatin abolished SET binding. ALDH2 and GSTP1 efficiency was assessed by enzymatic and comet assay. A lower ALDH2 activity was associated with greater DNA damage (tail intensity) in HEK293/SET compared with HEK293 cells, whereas HN13/siSET showed ALDH2 activity higher than HN13 cells. HN13/siSET cells showed increased tail intensity. Cisplatin-induced DNA damage response showed negative relationship between SET overexpression and BRCA2 recruitment. SET downregulated repair genes ATM, BRCA1 and CHEK2 and upregulated TP53. Cisplatin-induced cell-cycle arrest occurred in G(0) /G(1) and S in HEK293 cells, whereas HEK293/SET showed G(2) /M stalling. Overall, cisplatin was more cytotoxic for HN13 than HN13/siSET cells. Our data suggest a role for SET in cellular detoxification, DNA damage response and genome integrity.