ABSTRACT
In this work, we describe a novel ruthenium-xanthoxylin complex, [Ru(phen)2(xant)](PF6) (RXC), that can eliminate colorectal cancer (CRC) stem cells by targeting the chaperone Hsp90. RXC exhibits potent cytotoxicity in cancer cell lines and primary cancer cells, causing apoptosis in HCT116 CRC cells, as observed by cell morphology, YO-PRO-1/PI staining, internucleosomal DNA fragmentation, mitochondrial depolarization, and PARP cleavage (Asp214). Additionally, RXC can downregulate the HSP90AA1 and HSP90B1 genes and the expression of HSP90 protein, as well as the expression levels of its downstream/client elements Akt1, Akt (pS473), mTOR (pS2448), 4EBP1 (pT36/pT45), GSK-3ß (pS9), and NF-κB p65 (pS529), implying that these molecular chaperones can be molecular targets for RXC. Moreover, this compound inhibited clonogenic survival, the percentage of the CRC stem cell subpopulation, and colonosphere formation, indicating that RXC can eliminate CRC stem cells. RXC reduced cell migration and invasion, decreased vimentin and increased E-cadherin expression, and induced an autophagic process that appeared to be cytoprotective, as autophagy inhibitors enhanced RXC-induced cell death. In vivo studies showed that RXC inhibits tumor progression and experimental metastasis in mice with CRC HCT116 cell xenografts. Taken together, these results highlight the potential of the ruthenium complex RXC in CRC therapy with the ability to eliminate CRC stem cells by targeting the chaperone Hsp90.
Subject(s)
Colorectal Neoplasms , Ruthenium , Humans , Animals , Mice , Signal Transduction , Glycogen Synthase Kinase 3 beta/metabolism , HCT116 Cells , HSP90 Heat-Shock Proteins/metabolism , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Cell Proliferation , Cell Line, TumorABSTRACT
Ruthenium-based compounds have gained great interest due to their potent cytotoxicity in cancer cells; however, much of their potential applications remain unexplored. In this paper, we report the synthesis of a novel ruthenium complex with xanthoxylin (RCX) and the investigation of its cellular and molecular action in human hepatocellular carcinoma HepG2 cells. We found that RCX exhibited a potent cytotoxic effect in a panel of cancer cell lines in monolayer cultures and in a 3D model of multicellular cancer spheroids formed from HepG2 cells. This compound is detected at a high concentration in the cell nuclei, induces DNA intercalation and inhibits DNA synthesis, arresting the cell cycle in the S-phase, which is followed by the activation of the caspase-mediated apoptosis pathway in HepG2 cells. Gene expression analysis revealed changes in the expression of genes related to cell cycle control, apoptosis and the MAPK pathway. In addition, RCX induced the phosphorylation of ERK1/2, and pretreatment with U-0126, an MEK inhibitor known to inhibit the activation of ERK1/2, prevented RCX-induced apoptosis. In contrast, pretreatment with a p53 inhibitor (cyclic pifithrin-α) did not prevent RCX-induced apoptosis, indicating the activation of a p53-independent apoptosis pathway. RCX also presented a potent in vivo antitumor effect in C.B-17 SCID mice engrafted with HepG2 cells. Altogether, these results indicate that RCX is a novel anticancer drug candidate.
Subject(s)
Acetophenones/pharmacology , Apoptosis/drug effects , Cell Cycle Checkpoints/drug effects , MAP Kinase Signaling System/drug effects , Ruthenium/pharmacology , S Phase/drug effects , Tumor Suppressor Protein p53/metabolism , Acetophenones/chemical synthesis , Acetophenones/chemistry , Animals , Antineoplastic Agents/pharmacology , Caspase Inhibitors/pharmacology , Caspases/metabolism , DNA/biosynthesis , Female , Gene Expression Regulation, Neoplastic/drug effects , Hep G2 Cells , Humans , Intercalating Agents/pharmacology , Membrane Potential, Mitochondrial/drug effects , Mice, SCID , Models, Biological , Protein Kinase Inhibitors/pharmacology , Proton Magnetic Resonance Spectroscopy , Spheroids, Cellular/drug effects , Spheroids, Cellular/metabolism , Spheroids, Cellular/pathology , Xenograft Model Antitumor AssaysABSTRACT
The magnesium complex [Mg(hesp)2(phen)] (1), where hesp=hesperidin and phen=1,10'-phenanthroline, was synthesized and characterized by Elemental Analysis (C,H,N), atomic absorption and spectroscopic (FTIR, UV-visible, (1)H NMR) techniques. The congested structure facilitates the tilting and contact of the two hesperidin ligands by hydrogen bonding interactions having a stabilizer effect on the hesperidin. The hydrogen bonds are strongly affected by the solvent used which can lead to changes in the physical-chemical, luminescence and biologic properties of complex 1. Complex 1 is more hydrosoluble (S=472±3.05µgmL(-1)) and liposoluble (log P=-0.15±0.01) than free hesperidin (S=5.92±0.49µgmL(-1), log P=0.30). Oxidation of the complex in an aqueous solution and room temperature investigated by cyclic voltammetry resulted in a very stable two-electron cyclic process to form the phenoxonium neutral, cation and dication radicals. The stability of the voltammetric process indicates that the species produced are never exhausted and does not lead to changes in the coordination sphere composition. The complex was found to be a better radical scavenger for superoxide radical (IC50=68.3µM at pH7.8) than free hesperidin (IC50=116.68µmolL(-1)) and vitamin C (IC50=852µmolL(-1)). The strong blue fluorescence of complex 1 switches through loss of luminescence in pure water/protic organic solvents or when protected from water (in octanol for example as a model of phospholipid membranes). These features provide an opportunity to map the reactivity of hesperidin in the physiologic medium. In this context, a high uptake of complex into HeLa cells was detected by fluorescence microscopy. The blue fluorescence was uniformly distributed mainly in per nucleic region.