A series of
new polymetallic complexes have been prepared which couple
d6 pseudooctahedral lightabsorbing centers to a
d8 square planar platinum site. These systems have
lowest lying excited states that are M → BL charge transfer in
nature. Hence, optical excitation directs charge flow toward the
coupled platinum site. Synthetic variation of the lightabsorbing
metal and the bridging ligand has been quite useful in the
interpretation of the spectroscopic and electrochemical properties of
these systems.
A new type of mixed-metal, supramolecular complex has been designed that incorporates a platinum center to allow binding to DNA. The interaction of two such platinum heterobimetallic complexes of the general formula [(bpy)(2)M(dpb)PtCl(2)]Cl(2) (M = Ru(II), Os(II); bpy = 2,2'-bipyridine; dpb = 2,3-bis(2-pyridyl)benzoquinoxaline) with DNA is reported herein. The modular design of these systems allows for synthetic variation of individual components within this structural motif. In this case, the remote metal is varied from Ru(II) to Os(II). DNA binding was analyzed using non-denaturing agarose gel electrophoresis. The interaction of these complexes with DNA was studied relative to the known DNA cross-linkers, cis-[Pt(NH(3))Cl(2)] (cisplatin) and trans-{[PtCl(NH(3))(2)](2)(&mgr;-H(2)N(CH(2))(6)NH(2))}(2+) (1,1/t,t). Our mixed-metal Ru,Pt and Os,Pt compounds retard the migration of DNA through the gel in both a concentration- and time-dependent manner. Their effect on the migration of DNA is similar to, although much more dramatic than, that observed for either cisplatin or 1,1/t,t. Our evidence suggests a covalent binding of our mixed-metal complexes to DNA through the platinum site. The degree of retardation of DNA migration suggests a large change in DNA conformation is induced by binding of our mixed-metal complexes. This work establishes these inorganic systems as a new class of DNA-binding agents and lays the groundwork for future efforts to enhance binding in an effort to develop novel anticancer drugs through serial design and testing.
In this paper the characterization of the first near-infrared (NIR) phospholipase-activated molecular beacon is reported and its utility for in vivo cancer imaging is demonstrated. The probe consists of three elements: a phospholipid (PL) backbone to which the NIR fluorophore, pyropheophorbide a (Pyro), and the NIR Black Hole Quencher 3 (BHQ) were conjugated. Due to the close proximity of BHQ to Pyro, the Pyro-PtdEtn-BHQ probe is self-quenched until enzyme hydrolysis releases the fluorophore. The Pyro-PtdEtn-BHQ probe is highly specific to one isoform of phospholipase C, phosphatidylcholine-specific phospholipase C (PC-PLC), responsible for catabolizing phosphatidylcholine directly to phosphocholine. Incubation of Pyro-PtdEtn-BHQ in vitro with PC-PLC demonstrated a 150-fold increase in fluorescence that could be inhibited by the specific PC-PLC inhibitor tricyclodecan-9-yl xanthogenate (D609) with an IC50 of 34±8 µM. Since elevations in phosphocholine have been consistently observed by magnetic resonance spectroscopy in a wide array of cancer cells and solid tumors, we assessed the utility of Pyro-PtdEtn-BHQ as a probe for targeted tumor imaging. Injection of Pyro-PtdEtn-BHQ into mice bearing DU145 human prostate tumor xenografts followed by in vivo NIR imaging resulted in a 4-fold increase in tumor radiance over background and a 2 fold increase in the tumor:muscle ratio. Tumor fluorescence enhancement was inhibited with administration of D609. The ability to image PC-PLC activity in vivo provides a unique and sensitive method of monitoring one of the critical phospholipase signaling pathways activated in cancer, as well as the phospholipase activities that are altered in response to cancer treatment.
The effects of the selective peroxisome proliferator activated receptor-gamma (PPAR-γ) inhibitor GW9662 on phenylbutyrate (PB)-induced NMR-detectable lipid metabolites was investigated on DU145 prostate cancer cells. DU145 cells were perfused with 10 mM PB in the presence or absence of 1 µM of GW9662 and the results monitored by 31P and diffusion-weighted 1H NMR spectroscopy. GW9662 completely reversed PB-induced NMR-visible lipid and total choline accumulation in 1H spectra and glycerophosphocholine and β-NTP in 31P spectra. In addition, pre-incubation with GW9662 significantly reduced PB-induced caspase-3 activation, reversed the G1 block as measured by flow cytometry, and otherwise had little effect on cell survival as measured by MTT assay. These results suggest that the NMR visible lipid accumulation and apoptosis induced by PB treatment occurs through a mechanism that is mediated by PPAR-γ.
Saccharomyces cerevisiae responds to extracellular toxic stimuli by increasing intracellular cyclic AMP levels, leading to activation of a cAMP-dependent protein kinase, protein kinase A (PKA). Activated PKA phosphorylates downstream substrates, including specific DNA-binding proteins, to turn off the expression of most or all of the yeast genes. Such cAMP-PKA-mediated inhibition of gene expression in response to toxic stimuli appears to be unique to S. cerevisiae. For instance, in mammalian cells, the cAMP-PKA signaling pathway is rather responsive to growth factors and hormones in addition to being primarily involved in the activation of gene expression. Activation of gene expression by the cAMP-PKA pathway in mammalian cells is due mainly to the presence of cAMP-response elements (CREs) located in the promoters of many mammalian genes, and the expression of PKA-responsive stimulatory transcription factor CRE-binding protein, commonly referred as CREBP, which binds to the CREs. Thus, activation of the cAMP-PKA signaling pathway results in the phosphorylation of CREBP by PKA, and phosphorylated CREBP transactivates specific gene expression by interacting with the cognate CRE. Based on these findings, we sought to engineer a yeast-based biosensor, in which the stress-sensing cAMP-PKA pathway of yeast is coupled to the mammalian CREBP-CRE-stimulated gene expression pathway, which drives the expression of a reporter protein, such as green fluorescent protein (GFP). As a primary step toward the development of this biosensor, we engineered a yeast strain, BioS-1, by genetically altering YPH 501, a wild-type strain of S. cerevisiae, to express human CREBP and human CRE promoter-driven GFP. Exposure of BioS-1 to varying concentrations of As3+, Fe2+, Pb2+, and Cd2+ elicits concentration-dependent expression of the GFP reporter that can be easily monitored by the fluorescence emitted by GFP. The results also indicate that the engineered BioS-1 yeast cells can detect 2.5 ppm of these toxic metals and report it through the expression of GFP within 3 h. The results presented herein demonstrate that this engineered yeast strain can detect metal toxicants and can validate the use of this prototypic yeast strain to develop a biosensor that can be used to detect and monitor cytotoxic water-borne toxic heavy metals.
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