RESUMO
Hepatocellular carcinoma (HCC) is an essentially incurable inflammation-related cancer. We have previously shown by network analysis of proteomic data that the flavonoids epigallocatechin gallate (EGCG) and fisetin (FIS) efficiently downregulated pro-tumor cytokines released by HCC through inhibition of Akt/mTOR/RPS6 phospho-signaling. However, their mode of action at the global transcriptome level remains unclear. Herein, we endeavor to compare gene expression alterations mediated by these compounds through a comprehensive transcriptome analysis based on RNA-seq in HEP3B, a responsive HCC cell line, upon perturbation with a mixture of prototypical stimuli mimicking conditions of tumor microenvironment or under constitutive state. Analysis of RNA-seq data revealed extended changes on HEP3B transcriptome imposed by test nutraceuticals. Under stimulated conditions, EGCG and FIS significantly modified, compared to the corresponding control, the expression of 922 and 973 genes, respectively, the large majority of which (695 genes), was affected by both compounds. Hierarchical clustering based on the expression data of shared genes demonstrated an almost identical profile in nutraceutical-treated stimulated cells which was virtually opposite in cells exposed to stimuli alone. Downstream enrichment analyses of the co-modified genes uncovered significant associations with cancer-related transcription factors as well as terms of Gene Ontology/Reactome Pathways and highlighted ECM dynamics as a nodal modulation point by nutraceuticals along with angiogenesis, inflammation, cell motility and growth. RNA-seq data for selected genes were independently confirmed by RT-qPCR. Overall, the present systems approach provides novel evidence stepping up the mechanistic understanding of test nutraceuticals, thus rationalizing their clinical exploitation in new preventive/therapeutic modalities against HCC.
RESUMO
Modified purine derivatives exemplified by pyrazolopyrimidines have emerged as highly selective inhibitors of several angiogenic receptor tyrosine kinases. Herein, we designed and synthesized a new series of substituted pyrazolopyridines and explored their ability to influence crucial pro-angiogenic attributes of endothelial cells. Four of the synthesized compounds, possessing analogous substitution pattern, were found able to inhibit at low micromolar concentrations endothelial cell proliferation, migration and differentiation, constitutively or in response to Vascular Endothelial Growth Factor (VEGF) and to attenuate VEGF-induced phosphorylation of VEGF receptor-2 and downstream kinases AKT and ERK1/2. Administration of effective compounds in mice delayed the growth of syngeneic Lewis lung carcinoma transplants and reduced tumor microvessel density, without causing toxicity. Genome-wide microarray and gene ontology analyses of treated endothelial cells revealed derivative 18c as the most efficient modulator of gene expression and "mitotic cell cycle/cell division" along with "cholesterol biosynthesis" as the most significantly altered biological processes.