Compacting effect of BBR3464, a new-generation trisplatinum anticancer agent,on DNA

BBR3464 is a trinuclear platinum compound of formula [{trans-PtCl(NH₃)₂}₂-μ-trans-Pt(NH₃)₂{NH₂(CH₂)₆NH₂}₂]⁴⁺. It is a new-generation platinum chemotherapeutic agent that exhibits cytotoxicity at ten to thousand times lower dose limit compared to the well-known platinum drug cisplatin, in cisplatin-sensitive as well as in cisplatin-resistant cells. DNA is thought to be the primary cellular target of BBR3464. In this work, we have applied high-resolution atomic force microscopy (AFM) for the first time, to obtain direct information on BBR3464-induced structural changes of DNA. It is found that the DNA molecules get compacted after treatment with BBR3464, for the drug:DNA molar ratio and the drug treatment period of 0.01 and 48 h, respectively. These values of molar ratio and incubation period have been obtained previously, as a result of biochemical optimization studies carried out for achieving maximum drug effects. The DNA structural changes, as observed in AFM topographs, have been correlated to the bulk level spectroscopic information. A remark on the significance of BBR3464-induced DNA compaction with respect to the available AFM reports on DNA modification by cisplatin has been made.     

Differential recognition by the tumor suppressor protein p53 of DNA modified by the novel antitumor trinuclear platinum drug BBR3464 and cisplatin

The trinuclear platinum agent BBR3464, a representative of a new class of anticancer drugs, is more potent than conventional mononuclear cisplatin [cis-diamminedichloroplatinum(II)]. BBR3464 retains significant activity in human tumor cell lines and xenografts that are refractory or poorly responsive to cisplatin, and displays a high activity in human tumor cell lines that are characterized by both wild-type and mutant p53 gene. In contrast, on average, cells with mutant p53 are more resistant to the effect of cisplatin. It has been hypothesized that the sensitivity or resistance of tumor cells to cisplatin might be also associated with cell cycle control and repair processes that involve p53. DNA is a major pharmacological target of platinum compounds and DNA binding activity of the p53 protein is crucial for its tumor suppressor function. This study, using gel-mobility-shift assays, was undertaken to examine the interactions of active and latent p53 protein with DNA fragments and oligodeoxyribonucleotide duplexes modified by BBR3464 in a cell free medium and to compare these results with those describing the interactions of these proteins with DNA modified by cisplatin. The results indicate that structurally different DNA adducts of BBR3464 and cisplatin exhibit a different efficiency to affect the binding affinity of the modified DNA to p53 protein. It has been suggested that different structural perturbations induced in DNA by the adducts of BBR3464 and cisplatin produce a differential response to p53 protein activation and recognition and that a 'molecular approach' to control of downstream effects such as protein recognition and pathways of apoptosis induction may consist in design of structurally unique DNA adducts as cell signals.     

Structural changes of DNA induced by mono- and binuclear cancer drugs

The structural features of the drug-DNA adducts resulted from treatment of DNA with the platinum based mononuclear drug cisplatin and the binuclear drug [{trans-PtCl(NH3)2}2H2N(CH2)4NH2]Cl2 or bis(platin) have been investigated by atomic force microscopy (AFM). Reduction in the contour length of the DNA fragments has been observed after cisplatin treatment while, compaction and aggregation are found to be the primary structural modifications following treatment with the binuclear drug. The intermolecular interaction upon bis(platin) treatment leads to observation of highly condense aggregates without a distinct sight of single isolated DNA molecule. These differences in drug binding indicate that unlike the mononuclear drug cisplatin, bis(platin) causes extensive interhelical/intermolecular cross-linking through its multiple linking sites. To our knowledge, this is the first report of a comparative AFM study to monitor the effects of a mono- and a binuclear platinum anti-cancer drug on DNA structure. These observations should provide clues towards explaining the distinct biological activities of the two drugs.     

Study of the DNA/ethidium bromide interactions on mica surface by atomic force microscope: influence of the surface friction

The influence of mica surface on DNA/ethidium bromide interactions is investigated by atomic force microscopy (AFM). We describe the diffusion mechanism of a DNA molecule on a mica surface by using a simple analytical model. It appears that the DNA diffusion on a mica surface is limited by the surface friction due to the counterion correlations between the divalent counterions condensed on both mica and DNA surfaces. We also study the structural changes of linear DNA adsorbed on mica upon ethidium bromide binding by AFM. It turns out that linear DNA molecules adsorbed on a mica surface are unable to relieve the topological constraint upon ethidium bromide binding. In particular, strongly adsorbed molecules tend to be highly entangled, while loosely bound DNA molecules appear more extended with very few crossovers. Adsorbed DNA molecules cannot move freely on the surface because of the surface friction. Therefore, the topological constraint increases due to the ethidium bromide binding. Moreover, we show that ethidium bromide has a lower affinity for strongly bound molecules due to the topological constraint induced by the surface friction.     

SSB and the RecG DNA helicase: an intimate association to rescue a stalled replication fork

In E. coli, the regression of stalled DNA replication forks is catalyzed by the DNA helicase RecG. One means of gaining access to the fork is by binding to the single strand binding protein or SSB. This interaction occurs via the wedge domain of RecG and the intrinsically disordered linker (IDL) of SSB, in a manner similar to that of SH3 domains binding to PXXP motif‐containing ligands in eukaryotic cells. During loading, SSB remodels the wedge domain so that the helicase domains bind to the parental, duplex DNA, permitting the helicase to translocate using thermal energy. This translocation may be used to clear the fork of obstacles, prior to the initiation of fork regression.     

Design and synthesis of new antitumor agents with the 1,7-epoxycyclononane framework. Study of their anticancer action mechanism by a model compound

This article describes the design, synthesis and biological evaluation of a new family of antitumor agents having the 1,7-epoxycyclononane framework. We have developed a versatile synthetic methodology that allows the preparation of a chemical library with structural diversity and in good yield. The synthetic methodology has been scaled up to the multigram level and can be developed in an enantioselective fashion. The study in vitro of a model compound, in front of the cancer cell lines HL-60 and MCF-7, showed a growth inhibitory effect better than that of cisplatin. The observation of cancer cells by fluorescence microscopy showed the presence of apoptotic bodies and a degradation of microtubules. The study of cell cycle and mechanism of death of cancer cells by flow cytometry indicates that the cell cycle arrested at the G₀/G₁ phase and that the cells died by apoptosis preferably over necrosis. A high percentage of apoptotic cells at the subG₀/G₁ level was observed. This indicates that our model compound does not behave as an antimitotic agent like nocodazole, used as a reference, which arrests the cell cycle at G₂/M phase. The interaction of anticancer agents with DNA molecules was evaluated by atomic force microscopy, circular dichroism and electrophoresis on agarose gel. The results indicate that the model compound has not DNA as a target molecule. The in silico study of the model compound showed a potential good oral bioavailability.