Rapid DNA interstrand cross-linking of Pt(IV) compound.

Pt(IV) anticancer compounds have been developed for several decades to overcome the drawbacks of their Pt(II) congeners, and the reduction of Pt(IV) to Pt(II) has been commonly regarded as a necessary step in the activation of Pt(IV) compounds prior to targeting DNA. However, blockage of glutathione (GSH) biosynthesis resulted in a slight effect on the cytotoxicity of oxoplatin in yeast Saccharomyces cerevisiae strains, urging us to reconsider the mechanism of actions for the "inert" Pt(IV) complexes. Using X-ray absorption near-edge spectroscopy (XANES), our data demonstrated that Pt(IV) complex oxoplatin could bind to DNA in a tetravalent state. Both alkaline denaturing agarose electrophoresis and thermal denaturation-renaturation assay revealed that oxoplatin could rapidly produce stable interstrand crosslinks (ICLs), which can further translate into a fast cell-killing process in cancer cells. Using quantitative real-time PCR and immunofluorescence analysis, we also proved that Pt(IV) complex oxoplatin could induce a quick intracellular response of the FA/BRCA pathway in cancer cells that involves the DNA interstrand crosslinking repair system, and this quick response to ICLs was independent with the intracellular GSH levels. Cell cycle analysis showed that short incubation with oxoplatin can induce a strong S phase arrest in HeLa cells, indicating that the rapid interstrand crosslinks produced by oxoplatin might stall the replication fork, result in the double-strand breaks, and eventually induce cell death. Our results implied that, besides the reduction mechanism to release the Pt(II) congeners, direct and rapid interstrand cross-linking with DNA by Pt(IV) compounds might be a unique mechanism for Pt(IV) compounds, which may provide new insight for the development of next-generation platinum-based drugs.

Rapid induction of apoptosis in tumor cells treated with a new platinum(II) complex based on amino-thiazolidinone.

Thiazolidinone derivatives have been previously shown significant anti-cancer activities. Two amino-thiazolidinone complexes, [Pt(HTone)Cl] (1) and [Cu(HTone)Cl] (3) (HTone = (Z)-2-((E)-(1-(pyridin-2-yl)ethylidene)hydrazono)thiazolidin-4-one) and one ethyl-modified [Pt(ETone)Cl2] (2) (ETone = (Z)-3-ethyl-2-((E)-(1-(pyridin-2-yl)ethylidene) hydrazono)thiazolidin-4-one)], were designed and synthesized in order to explore novel metal-based antitumor agents. MTT assay indicated that 1 and 3 were markedly cytotoxic to MCF-7, HepG-2 and NCI-H460 tumor cells, superior to both cisplatin and the HTone ligand. Massive dead cells were observed as early as 6 h when treated with 1, indicating rapid action of 1 as compared to that of other compounds. More interestingly, Hoechst 33342 staining and flow cytometry analysis illustrated that only complex 1 could induce obvious cell apoptosis within 12 h, which was associated with the high-expression of Bax and cleavage of caspase-3 from 35 kDa to 17 kDa. By means of ICP-MS assay, we found complex 1 could largely accumulate in tumor cells in a short time. Additionally, complex 1 showed no cross resistance against the cisplatin-resistant cells.

Four Cu(ii) complexes based on antitumor chelators: synthesis, structure, DNA binding/damage, HSA interaction and enhanced cytotoxicity.

Four novel copper(ii) complexes [Cu(II)(Bp4mT)(µ-Cl)]2 (), [Cu(II)(µ-Bp4mT)Br]2 (), [Cu(II)(HBpT)Cl] (), and [Cu(II)(HBpT)Br] () (Bp4mT = 2-benzoylpyridine-4-methylthiosemicarbazone, HBpT = 2-benzoylpyridine thiosemicarbazone), were synthesized and characterized using single-crystal X-ray diffraction, elemental analysis, infrared, and ultraviolet-visible spectroscopy. X-ray analysis revealed that complexes and based on the Bp4mT ligand presented dimeric structures in which the Cu(ii) ions were located in a five-coordinated distorted square-pyramidal geometry, whereas both and complexes were mononuclear with the Cu(ii) ions exhibiting a tetracoordinated square planar configuration. Their interactions with calf thymus DNA (CT-DNA) were investigated using viscosity measurements and fluorescence spectroscopy. Multispectroscopic evidence has shown interactions between these complexes and human serum albumin (HSA). All these complexes have exhibited efficient oxidative cleavage of supercoiled DNA in the presence of hydrogen peroxide, presumably via an oxidative mechanism. Furthermore, in vitro cytotoxicity studies of against human liver hepatocellular carcinoma cells (HepG-2), human large cell lung carcinoma cells (NCI-H460), and human cervical carcinoma cells (HeLa) indicated their promising antitumor activity with quite low IC50 values in the range of 0.08-1.98 µM, which are 83 times lower than those of cisplatin. The mechanistic studies revealed that four complexes, which induced early apoptosis, were involved in reactive oxygen species generation and DNA cleavage for their antitumor activities.

Association of structural modifications with bioactivity in three new copper(II) complexes of Schiff base ligands derived from 5-chlorosalicylaldehyde and amino acids.

Three novel structurally associated copper(II) complexes [Cu(II)(SalCl-Gly)(H2O)2] (1), [Cu(II)(SalCl-Ala)(H2O)] (2) and [Cu(II)(SalCl-Gly)(bipy)]·0.5H2O (3) (SalCl-Gly=5-chloro-2-hydroxybenzylidene-glycine, SalCl-Ala=5-chloro-2-hydroxybenzylidene-alanine, bipy=2,2'-bipyridine) have been synthesized and characterized by X-ray crystallography, elemental analysis, IR and fluorescence spectroscopy. Single-crystal diffraction reveals that complex 1 is an infinite 1D zigzag chain in which SalCl-Gly serves as both a chelating and a bridging ligand, while complexes 2 and 3 are mononuclear. Cu(II) ions in complexes 1-3 exhibit distorted quasi-hexacoordinated octahedral, tetracoordinated square planar, and pentacoordinated square pyramid geometry, respectively. Their interactions with calf thymus DNA (CT-DNA) have been investigated by viscosity measurements and fluorescence spectroscopy. The apparent binding constant (Kapp) values for 1-3 are 1.02×10(5), 0.98×10(5) and 1.57×10(5)M(-1), respectively. All complexes displayed efficient oxidative cleavage of supercoiled DNA in the presence of H2O2. Complex 2, whose ligand can be regarded as a methyl-modification of SalCl-Gly of 1, showed a reduced DNA cleavage activity and a little-changed DNA-binding ability compared with 1. While attaching a 2,2'-bipyridine group to 1, the resulting complex 3 was conferred an enhanced intercalation into DNA. Moreover, cytotoxicity studies of three complexes against HepG-2 (human liver hepatocellular carcinoma) and NCI-H460 (human large-cell lung carcinoma) cells indicated that, thereto, complex 3 possessed the highest inhibition on viability of tested cells.

Functional study of the Hap4-like genes suggests that the key regulators of carbon metabolism HAP4 and oxidative stress response YAP1 in yeast diverged from a common ancestor.

The transcriptional regulator HAP4, induced by respiratory substrates, is involved in the balance between fermentation and respiration in S. cerevisiae. We identified putative orthologues of the Hap4 protein in all ascomycetes, based only on a conserved sixteen amino acid-long motif. In addition to this motif, some of these proteins contain a DNA-binding motif of the bZIP type, while being nonetheless globally highly divergent. The genome of the yeast Hansenula polymorpha contains two HAP4-like genes encoding the protein HpHap4-A which, like ScHap4, is devoid of a bZIP motif, and HpHap4-B which contains it. This species has been chosen for a detailed examination of their respective properties. Based mostly on global gene expression studies performed in the S. cerevisiae HAP4 disruption mutant (ScΔhap4), we show here that HpHap4-A is functionally equivalent to ScHap4, whereas HpHap4-B is not. Moreover HpHAP4-B is able to complement the H2O2 hypersensitivity of the ScYap1 deletant, YAP1 being, in S. cerevisiae, the main regulator of oxidative stress. Finally, a transcriptomic analysis performed in the ScΔyap1 strain overexpressing HpHAP4-B shows that HpHap4-B acts both on oxidative stress response and carbohydrate metabolism in a manner different from both ScYap1 and ScHap4. Deletion of these two genes in their natural host, H. polymorpha, confirms that HpHAP4-A participates in the control of the fermentation/respiration balance, while HpHAP4-B is involved in oxidative stress since its deletion leads to hypersensitivity to H2O2. These data, placed in an evolutionary context, raise new questions concerning the evolution of the HAP4 transcriptional regulation function and suggest that Yap1 and Hap4 have diverged from a unique regulatory protein in the fungal ancestor.

Nuclease activity and protein-binding properties of a novel tetranuclear thiosemicarbazide Pt(II) complex.

A novel tetranuclear [Pt4(Am4M)4]·16.25H2O (1), where H2Am4M is the thiosemicarbazone ligand (Z)-2-(amino(pyridin-2-yl)methylene)-N-methylhydrazinecarbothioamide, has been synthesized and characterized by using various physico-chemical techniques. X-ray analysis revealed that the Pt(II) complex consists of a neutral tetranuclear [Pt4(Am4M)4] unit and abundant water molecules. The tetranuclear unit is stabilized by strong intramolecular π-π stacking and thus presents a ship-like eight-membered ring [Pt4S4] with four tridentate ligands peripherally coordinated to four Pt(II) ions, constituting a novel type of "well" structure. DNA and protein binding properties of complex 1 have been studied by UV and fluorescence spectroscopic methods. The binding constant (Kb = 3.05 × 10(6) M(-1)) and the apparent binding constant (Kapp = 7.732 × 10(6) M(-1)) to calf thymus DNA (CT-DNA) were accurately fitted by two respective non-linear equations, suggesting an efficient DNA intercalative binding mode which was confirmed by viscometric experiments. Furthermore, following agarose gel electrophoresis experiments, we observed that the plasmid DNA pUC19 could be completely digested by complex 1, depending on incubation time and concentration, indicating that complex 1 may possess an effective nuclease activity on DNA. Singlet oxygen ((1)O2) inhibitors showed an inhibitory effect on the cleavage. Fluorescence spectrometry also detected a medium affinity of complex 1 to bovine serum albumin (BSA) at different temperatures, which was tentatively assigned to a static binding mode. All these results suggested that this poly-Pt(II) complex might exhibit biological action as a potential chemotherapy agent.

Biological evaluation of a cytotoxic 2-substituted benzimidazole copper(II) complex: DNA damage, antiproliferation and apoptotic induction activity in human cervical cancer cells.

Exploring novel chemotherapeutic agents is a great challenge in cancer medicine. To that end, 2-substituted benzimidazole copper(II) complex, [Cu(BMA)Cl2]·(CH3OH) (1) [BMA = N,N'-bis(benzimidazol-2-yl-methyl)amine], was synthesized and its cytotoxicity was characterized. The interaction between complex 1 and calf thymus DNA was detected by spectroscopy methods. The binding constant (K b = 1.24 × 10(4 )M(-1)) and the apparent binding constant (K app = 6.67 × 10(6 )M(-1)) of 1 indicated its moderate DNA affinity. Complex 1 induced single strand breaks of pUC19 plasmid DNA in the presence of H2O2 through an oxidative pathway. Cytotoxicity studies proved that complex 1 could inhibit the proliferation of human cervical carcinoma cell line HeLa in both time- and dose-dependent manners. The results of nuclei staining by Hoechst 33342 and alkaline single-cell gel electrophoresis proved that complex 1 caused cellular DNA damage in HeLa cells. Furthermore, treatment of HeLa cells with 1 resulted in S-phase arrest, loss of mitochondrial potential, and up-regulation of caspase-3 and -9 in HeLa cells, suggesting that complex 1 was capable of inducing apoptosis in cancer cells through the intrinsic mitochondrial pathway.

Gene responses to oxygen availability in Kluyveromyces lactis: an insight on the evolution of the oxygen-responding system in yeast.

The whole-genome duplication (WGD) may provide a basis for the emergence of the very characteristic life style of Saccharomyces cerevisiae-its fermentation-oriented physiology and its capacity of growing in anaerobiosis. Indeed, we found an over-representation of oxygen-responding genes in the ohnologs of S. cerevisiae. Many of these duplicated genes are present as aerobic/hypoxic(anaerobic) pairs and form a specialized system responding to changing oxygen availability. HYP2/ANB1 and COX5A/COX5B are such gene pairs, and their unique orthologs in the 'non-WGD' Kluyveromyces lactis genome behaved like the aerobic versions of S. cerevisiae. ROX1 encodes a major oxygen-responding regulator in S. cerevisiae. The synteny, structural features and molecular function of putative KlROX1 were shown to be different from that of ROX1. The transition from the K. lactis-type ROX1 to the S. cerevisiae-type ROX1 could link up with the development of anaerobes in the yeast evolution. Bioinformatics and stochastic analyses of the Rox1p-binding site (YYYATTGTTCTC) in the upstream sequences of the S. cerevisiae Rox1p-mediated genes and of the K. lactis orthologs also indicated that K. lactis lacks the specific gene system responding to oxygen limiting environment, which is present in the 'post-WGD' genome of S. cerevisiae. These data suggested that the oxygen-responding system was born for the specialized physiology of S. cerevisiae.

Oxygen-dependent transcriptional regulator Hap1p limits glucose uptake by repressing the expression of the major glucose transporter gene RAG1 in Kluyveromyces lactis.

The HAP1 (CYP1) gene product of Saccharomyces cerevisiae is known to regulate the transcription of many genes in response to oxygen availability. This response varies according to yeast species, probably reflecting the specific nature of their oxidative metabolism. It is suspected that a difference in the interaction of Hap1p with its target genes may explain some of the species-related variation in oxygen responses. As opposed to the fermentative S. cerevisiae, Kluyveromyces lactis is an aerobic yeast species which shows different oxygen responses. We examined the role of the HAP1-equivalent gene (KlHAP1) in K. lactis. KlHap1p showed a number of sequence features and some gene targets (such as KlCYC1) in common with its S. cerevisiae counterpart, and KlHAP1 was capable of complementing the hap1 mutation. However, the KlHAP1 disruptant showed temperature-sensitive growth on glucose, especially at low glucose concentrations. At normal temperature, 28 degrees C, the mutant grew well, the colony size being even greater than that of the wild type. The most striking observation was that KlHap1p repressed the expression of the major glucose transporter gene RAG1 and reduced the glucose uptake rate. This suggested an involvement of KlHap1p in the regulation of glycolytic flux through the glucose transport system. The DeltaKlhap1 mutant showed an increased ability to produce ethanol during aerobic growth, indicating a possible transformation of its physiological property to Crabtree positivity or partial Crabtree positivity. Dual roles of KlHap1p in activating respiration and repressing fermentation may be seen as a basis of the Crabtree-negative physiology of K. lactis.

Why do some yeast species require niacin for growth? Different modes of NAD synthesis.

NAD holds a key position in metabolism and cellular regulatory events as a major redox carrier and a signalling molecule. NAD biosynthesis pathways have been reconstructed and compared in seven yeast species with completely sequenced genomes, including Saccharomyces cerevisiae, Kluyveromyces lactis, Candida glabrata, Debaryomyces hansenii, Candida albicans, Yarrowia lipolytica and Schizosaccharomyces pombe. Both amino acid and nucleotide sequence similarity analysis in silico indicated that de novo NAD biosynthesis might not exist in K. lactis, C. glabrata and Schiz. pombe, while other species have the kynurenine pathway. It also showed that the NAD salvage pathway via nicotinic acid and nicotinic acid mononucleotide is conserved in all of these yeasts. Deletion of KlNPT1 (the gene for nicotinate phosphoribosyl-transferase) is lethal, which demonstrates that this salvage pathway, utilizing exogenous nicotinic acid, is the unique route to synthesize NAD in K. lactis. The results suggested that the basis of the variation of niacin requirements in yeasts lies in their different combinations of NAD biosynthesis pathways. The de novo pathway is absent but the salvage pathway is conserved in niacin-negative yeasts, while both pathways coexist in niacin-positive yeasts.

Cloning of the LAC4 Promoter from K. lactis and Studies on the Function of its UASII.

The -661 - +21 bp region of the LAC4 gene form K. lactis CBS141 was obtained by PCR technique and fused with the lacZ gene from E. coli to construct and expression vector YFD 114. It was then transformed into K. lactis Y167. The function of the cloned LAC4 promoter was tested by induction with galactose, lactose, sorbitol or IPTG. The results revealed that galactose had higher inducing effect than lactose while sorbitol and IPTG had no inducing effect. The insertion of chemically synthesized LAC4 UASII at just the upstream of the cloned LAC4 promoter could increase its basic and induced expression level. The extents of increase were different with the orientation and the copy number of the inserted UASII.