The mechanism of action of platinum(IV) complexes in ovarian cancer cell lines

The reduction potentials, lipophilicities, cellular uptake and cytotoxicity have been examined for two series of platinum(IV) complexes that yield common platinum(II) complexes on reduction: cis-[PtCl(4)(NH(3))(2)], cis,trans,cis-[PtCl(2)(OAc)(2)(NH(3))(2)], cis,trans,cis-[PtCl(2)(OH)(2)(NH(3))(2)], [PtCl(4)(en)], cis,trans-[PtCl(2)(OAc)(2)(en)] and cis,trans-[PtCl(2)(OH)(2)(en)] (en=ethane-1,2-diamine, OAc=acetate). As previously reported, the reduction occurs most readily when the axial ligand is chloride and least readily when it is hydroxide. The en series of complexes are marginally more lipophilic than their ammine analogues. The presence of axial chloride or acetate ligands results in a slighter higher lipophilicity compared with the platinum(II) analogue whereas hydroxide ligands lead to a substantially lower lipophilicity. The cellular uptake is similar for the platinum(II) species and their analogous tetrachloro complexes, but is substantially lower for the acetato and hydroxo complexes, resulting in a correlation with the reduction potential. The activities are also correlated with the reduction potentials with the tetrachloro complexes being the most active of the platinum(IV) series and the hydroxo being the least active. These results are interpreted in terms of reduction, followed by aquation reducing the amount of efflux from the cells resulting in an increase in net uptake.     

Cytotoxic activity of platinum (II) complexes with tri-n-butylphosphine. Crystal structure of the dinuclear hydrazine-bridged complex, cis,cis-[PtCl(PBu3n)2(mu-N2H4)PtCl(PBu3n) 2] (ClO4)2.2CHCl3.

A series of platinum(II) tri-n-butylphosphine complexes having the formulas cis-[PtCl2L2], NEt4[PtCl3L], [PtCl(en)L]Cl, [Pt(en)L2](ClO4)2, sym-trans-[Pt2Cl4L2], [Pt2Cl2L4](ClO4)2, trans,trans-[PtCl2L(mu-N2H4)PtCl2L] trans,trans-[PtCl2L(mu-en)PtCl2L], and cis,cis-[PtClL2(mu-N2H4)PtClL2](ClO4)2 (L = tri-n-butylphosphine; en = ethylenediamine) have been synthesized and their cytotoxic activity in vitro and in vivo has been studied. The solution behavior of the novel dinuclear diamine-bridged platinum(II) complexes has been investigated by means of UV and 31P NMR spectroscopy. For the ionic hydrazine compound cis,cis-[PtClL2(mu-N2H4)PtClL2](ClO4)2, an x-ray structure determination is reported. Crystal data: space group P2(1)/a, a = 17.803(1), b = 18.888(3), c = 12.506(3) A, beta = 107.97(2) degrees, Z = 2, R = 0.052, RW = 0.058. The platinum coordination is approximately square-planar, with the bond lengths Pt-Cl = 2.358(5), Pt-N = 2.15(1), Pt-P(trans to Cl) = 2.260(5), and Pt-P(trans to N) = 2.262(6) A. All investigated compounds were cytotoxic in vitro against L1210 cells and showed no cross-resistance to cisplatin. On the other hand, no antitumor activity was observed vs L1210 leucemia in DBA2 mice.     

The fate of platinum(II) and platinum(IV) anti-cancer agents in cancer cells and tumours

SRIXE mapping has been used to gain insight into the fate of platinum(II) and platinum(IV) complexes in cells and tumours treated with anticancer active complexes to facilitate the development of improved drugs. SRIXE maps were collected of thin sections of human ovarian (A2780) cancer cells treated with bromine containing platinum complexes, cis-[PtCl(2)(3-Brpyr)(NH(3))] (3-Brpyr=3-bromopyridine) and cis,trans,cis-[PtCl(2)(OAcBr)(2)(NH(3))(2)] (OAcBr=bromoacetate), or a platinum complex with an intercalator attached cis-[PtCl(2)(2-[(3-aminopropyl)amino]-9,10-anthracenedione)(NH(3))]. After 24h the complexes appear to be localised in the cell nucleus with a lower concentration in the surrounding cytoplasm. In cells treated with cis-[PtCl(2)(3-Brpyr)(NH(3))] the concentration of bromine was substantially higher than in control cells and the bromine was co-localised with the platinum consistent with the 3-bromopyridine ligand remaining bound to the platinum. The cells treated with cis,trans,cis-[PtCl(2)(OAcBr)(2)(NH(3))(2)] also showed an increased level of bromine, but to a much lesser extent than for those treated with cis-[PtCl(2)(3-Brpyr)(NH(3))] suggestive of substantial reduction of the platinum(IV) complex. Maps were also collected from thin sections of a 4T1.2 neo 1 mammary tumour xenograft removed from a mouse 3h after treatment with cis,trans,cis-[PtCl(2)(OH)(2)(NH(3))(2)] and revealed selective uptake of platinum by one cell.     

DNA-protein cross-linking by trans-[PtCl(2)(E-iminoether)(2)]. A concept for activation of the trans geometry in platinum antitumor complexes

The structure-pharmacological activity relationships generally accepted for antitumor platinum compounds stressed the necessity for the cis-[PtX(2)(amine)(2)] structure while the trans-[PtX(2)(amine)(2)] structure was considered inactive. However, more recently, several trans-platinum complexes have been identified which are potently toxic, antitumor-active and demonstrate activity distinct from that of conventional cisplatin (cis-[PtCl(2)(NH(3))(2)]). We have shown in the previous report that the replacement of ammine ligands by iminoether in transplatin (trans-[PtCl(2)(NH(3))(2)]) results in a marked enhancement of its cytotoxicity so that it is more cytotoxic than its cis congener and exhibits significant antitumor activity, including activity in cisplatin-resistant tumor cells. In addition, we have also shown previously that this new trans compound (trans-[PtCl(2)(E-iminoether)(2)]) forms mainly monofunctional adducts at guanine residues on DNA, which is generally accepted to be the cellular target of platinum drugs. In order to shed light on the mechanism underlying the antitumor activity of trans-[PtCl(2)(E-iminoether)(2)] we examined oligodeoxyribonucleotide duplexes containing a single, site-specific, monofunctional adduct of this transplatin analog by the methods of molecular biophysics. The results indicate that major monofunctional adducts of trans-[PtCl(2)(E-iminoether)(2)] locally distort DNA, bend the DNA axis by 21 degrees toward the minor groove, are not recognized by HMGB1 proteins and are readily removed from DNA by nucleotide excision repair (NER). In addition, the monofunctional adducts of trans-[PtCl(2)(E-iminoether)(2)] readily cross-link proteins, which markedly enhances the efficiency of this adduct to terminate DNA polymerization by DNA polymerases in vitro and to inhibit removal of this adduct from DNA by NER. It is suggested that DNA-protein ternary cross-links produced by trans-[PtCl(2)(E-iminoether)(2)] could persist considerably longer than the non-cross-linked monofunctional adducts, which would potentiate toxicity of this antitumor platinum compound toward tumor cells sensitive to this drug. Thus, trans-[PtCl(2)(E-iminoether)(2)] represents a quite new class of platinum antitumor drugs in which activation of trans geometry is associated with an increased efficiency to form DNA-protein ternary cross-links thereby acting by a different mechanism from 'classical' cisplatin and its analogs.     

Further studies on toxic and radiosensitizing properties of ruthenium complexes of 4-nitroimidazoles

Transition metal complexes containing nitroimidazole ligands have been shown previously to act as radiosensitizers of hypoxic cells in vitro. As part of our study on metal-radiosensitizer complexes, we were encouraged by a ruthenium (Ru) sensitizer, RuCl2(DMSO)2(4(5)-nitroimidazole)2, 1, which showed better radiosensitizing properties and lower toxicity than the free ligand. In this study, we have extended our investigation to include the various other substituted 4-nitroimidazoles as ligands. The new Ru complexes, analogues of 1, were synthesized, identified and characterized and their toxicity and radiosensitizing abilities examined in vitro using Chinese hamster ovary (CHO) cells. Like 1, each of these ruthenium complexes has lower CHO hypoxic toxicity than the free ligands alone at equimolar concentration. These newer complexes gave sensitizing enhancement ratio (SER) values of 1.1 to 1.3 at 1.0 X 10(-4) mol dm-3 compared with 1.6 for 1. Unlike complex 1, the new complexes do not bind to plasmid DNA (assessed by inhibition of restriction endonuclease activity), possibly because the chloride (Cl-) ligand does not dissociate. In addition, the redox potential of the coordinated imidazole ligands is relatively unchanged compared to that of the free ligand. These factors may explain the more favourable properties of 1 compared with those of the new 4-nitroimidazole complexes of Ru.     

A circular dichroism study of DNA.platinum complexes. Differentiation between monofunctional, cis-bidentate and trans-bidentate platinum fixation on a series of DNAs

The circular dichroism (CD) spectra of a series of DNA . platinum complexes are presented. The following platinum compounds, [Pt(dien)Cl]Cl, cis-Pt(NH3)2Cl2, cis-Pt(en)Cl2, trans-Pt-(NH3)2Cl2, K[Pt(NH3)Cl3] and K2[PtCl4] were complexed with the DNA extracted from bacteria Micrococcus lysodeikticus (72% dG + dC), Escherichia coli (50% dG + dC), Clostridium perfringens (32% dG + dC) and salmon sperm (41% dG + dC). Strong differences were found between the different DNA . Pt complexes. Three types of spectra clearly demonstrate the different platinum binding modes on DNA. In the first type, the platinum compound, i.e. [Pt(dien)Cl]Cl, is fixed to DNA with only one bond (monofunctional complex formation) and no significant change of the CD positive band of DNA is found. The main feature of the second type is a continuous intensity decrease of the positive band as observed for trans-Pt(NH3)2Cl2 (trans-bidentate complex formation). The third type concerns the cis-bidentate platinum fixation obtained with cis-Pt(NH3)2Cl2, cis-Pt(en)Cl2, K[Pt(NH3)Cl3] and K2[PtCl4]. The CD spectra are in this case characterized by an increase in the positive Cotton effect which is dG + dC-dependent up to an rb value around 0.10 (where rb = number of platinum atoms bound per nucleotide), followed by a decrease until DNA saturation with platinum is reached. A linear decrease in the amplitude of the negative band is detected in all the complexes except in the case of the monofunctional DNA . Pt complexes. For the cis-bidentate and trans-bidentate platinum fixation, a continuous bathochromic shift occurs.     

DNA binding by antitumor trans-[PtCl2(NH3)(thiazole)]. Protein recognition and nucleotide excision repair of monofunctional adducts

Antitumor effects of cis-diamminedichloroplatinum(II) (cisplatin) and the clinical inactivity of its trans isomer (transplatin) have been considered a paradigm for the classical structure-activity relationships of platinum drugs. However, several new analogues of transplatin which exhibit a different spectrum of cytostatic activity including activity in tumor cells resistant to cisplatin have been recently identified. Analogues containing the planar amine ligand of the general structure trans-[PtCl(2)(NH(3))(L)], where L = planar amine, represent an example of such compounds. DNA is believed to be the major pharmacological target of platinum compounds. To contribute to the understanding of mechanisms underlying the activation of trans geometry in transplatin analogues containing planar amine ligands, various biochemical and biophysical methods were employed in previous studies to analyze the global modifications of natural DNA by trans-[PtCl(2)(NH(3))(L)]. These initial studies have revealed some unique features of the DNA binding mode of this class of platinum drugs. As the monofunctional lesions represent a significant fraction of stable adducts formed in DNA by bifunctional antitumor trans-platinum compounds with planar ligands, we analyzed in the present work short DNA duplexes containing the single, site-specific monofunctional adduct of a representative of this class of platinum drugs, antitumor trans-[PtCl(2)(NH(3))(thiazole)]. It has been shown that, in contrast to the adducts of monodentate chlorodiethylenetriamineplatinum(II) chloride or [PtCl(NH(3))(3)]Cl, the monofunctional adduct of trans-[PtCl(2)(NH(3))(thiazole)] inhibits DNA synthesis and creates a local conformational distortion similar to that produced in DNA by the major 1,2-GG intrastrand CL of cisplatin, which is considered the lesion most responsible for its anticancer activity. In addition, the monofunctional adducts of trans-[PtCl(2)(NH(3))(thiazole)] are recognized by HMGB1 domain proteins and removed by the nucleotide excision repair system similarly as the 1,2-GG intrastrand CL of cisplatin. The results of the present work further support the view that the simple chemical modification of the structure of an inactive platinum compound alters its DNA binding mode into that of an active drug and that processing of the monofunctional DNA adducts of the trans-platinum analogues in tumor cells may be similar to that of the major bifunctional adducts of "classical" cisplatin.     

DNA-targeted 2-nitroimidazoles: studies of the influence of the phenanthridine-linked nitroimidazoles, 2-NLP-3 and 2-NLP-4, on DNA damage induced by ionizing radiation

The nitroimidazole-linked phenanthridines 2-NLP-3 (5-[3-(2-nitro-1-imidazoyl)-propyl]-phenanthridinium bromide) and 2-NLP-4 (5-[3-(2-nitro-1-imidazoyl)-butyl]-phenanthridinium bromide) are composed of the radiosensitizer, 2-nitroimidazole, attached to the DNA intercalator phenanthridine by a 3- and 4-carbon linker, respectively. Previous in vitro assays showed both compounds to be 10-100 times more efficient as hypoxic cell radiosensitizers (based on external drug concentrations) than the untargeted 2-nitroimidazole radiosensitizer, misonidazole (Cowan et al., Radiat. Res. 127, 81-89, 1991). Here we have used a (32)P postlabeling assay and 5'-end-labeled oligonucleotide assay to compare the radiation-induced DNA damage generated in the presence of 2-NLP-3, 2-NLP-4, phenanthridine and misonidazole. After irradiation of the DNA under anoxic conditions, we observed a significantly greater level of 3'-phosphoglycolate DNA damage in the presence of 2-NLP-3 or 2-NLP-4 compared to irradiation of the DNA in the presence of misonidazole. This may account at least in part for the greater cellular radiosensitization shown by the nitroimidazole-linked phenanthridines over misonidazole. Of the two nitroimidazole-linked phenanthridines, the better in vitro radiosensitizer, 2-NLP-4, generated more 3'-phosphoglycolate in DNA than did 2-NLP-3. At all concentrations, phenanthridine had little effect on the levels of DNA damage, suggesting that the enhanced radiosensitization displayed by 2-NLP-3 and 2-NLP-4 is due to the localization of the 2-nitroimidazole to the DNA by the phenanthridine substituent and not to radiosensitization by the phenanthridine moiety itself.     

Comparison of chemical reactivity, cytotoxicity, interstrand cross-linking and DNA sequence specificity of bis(platinum) complexes containing monodentate or bidentate coordination spheres with their monomeric analogues

The properties of a new bis(platinum) complex containing two monodentate coordination spheres, [(trans-PtCl(NH3)2)2H2N(CH2)4NH2]Cl2 (1,1/t,t), are reported. Comparison is made with respect to chemical reactivity, in vitro biological activity in murine and tumor cells, DNA conformational changes, cross-linking efficiency, and sequence specificity between this complex and the previously reported complex containing two bidentate platinum atoms, [(Pt(mal)(NH3))2H2N(CH2)4NH2] (2,2/c,c), as well as with their respective monomeric analogues, [PtCl(dien)]Cl and cis-[PtCl2(NH3)2](cis-DDP). While both bis(platinum) complexes are active against cis-DDP-resistant cells, the monodentate bis(platinum) complex (1,1/t,t) has a lower resistance factor than the complex with bidentate coordination spheres (2,2/c,c). More importantly, this property is repeated in a human ovarian carcinoma cell line. DNA-binding studies show that DNA interstrand cross-linking is more efficient for the 1,1/t,t complex. DNA sequencing studies employing the exonuclease activity of T4-polymerase demonstrate that there are a variety of binding sites; some are common to all complexes and some common to both bis(platinum) complexes, while the monodentate 1,1/t,t species also reacts at unique sites, not attacked by any of the other complexes studied. The circular dichroism of CT DNA modified by the 1,1/t,t complex is also unique and is not seen for any of the other agents.     

Multinuclear NMR spectroscopy and antitumor activity of novel platinum(II) complexes with 5,7-disubstituted-1,2,4-triazolo[1,5-a]pyrimidines

Novel platinum(II) complexes with 5,7-disubstituted-1,2,4-triazolo[1,5-a]pyrimidines have been synthesized and characterized by infrared and multinuclear magnetic resonance spectroscopic techniques (1H, 13C, 15N, 195Pt). The complexes are of two types: [PtCl2(L)2] and [PtCl2(NH3)(L)], where L=5,7-diphenyl-1,2,4-triazolo[1,5-a]pyrimidine (dptp) and 5,7-ditertbutyl-1,2,4-triazolo[1,5-a]pyrimidine (dbtp). Significant 15N NMR upfield shifts (92-95 ppm) were observed for N(3) atom indicating this nitrogen atom as a coordination site. The molecular structure suggest that Pt(II) ion has the square planar geometry with N(3) bonded 5,7-disubstituted-1,2,4-triazolo[1,5-a]pyrimidines, N-bonded second ligand (NH3 for cis-[PtCl2(NH3)(L)] or, respectively, 5,7-disubstituted-1,2,4-triazolo[1,5-a]pyrimidines for cis-[PtCl2L2]) and two cis chloride anions. The antiproliferative activity in vitro of complexes (1-4) have been tested against the cells of four human cell lines: SW707 rectal adenocarcinoma, A549 non-small cell lung carcinoma, T47D breast cancer and HCV29T bladder cancer. The results indicate a moderate antiproliferative activity of (4) against the cells of rectal, breast and bladder cancer and a marked and selective cytotoxic effect of (1-3) against the cells of all studied human cancer lines.     

A study of interactions of platinum (II) compounds with DNA by means of CD spectra of solutions and liquid crystalline microphases of DNA

The optical properties of the DNA complexes with divalent platinum compounds of the cis-diamine type differing both in the nature of anionic and neutral ligands and in the spatial arrangement about the platinum atom were studied. The platinum compounds cis-[Pt(NH3)2Cl2], [Pt(en)Cl2], [Pt(tetrameen)Cl2], cis-[Pt(NH3)2NO2Cl], and cis-[PtNH3(Bz)Cl2] at small values of r (r is the molar ratio of a platinum compound to DNA nucleotides in the reaction mixture) were found to induce an increase in the amplitude of the positive band in the circular dichroic (CD) spectrum of linear DNA. All the compounds listed except cis-[Pt(NH3)2NO2Cl] caused a sharp decrease of the amplitude of the negative band in the CD spectrum of a liquid crystalline microphase of DNA formed in solution in the presence of poly(ethylene glycol). All these platinum compounds (except [Pt(tetrameen)Cl2]) exhibit biological (antimitotic, antitumour, etc.) activity. The platinum compounds trans-[Pt(NH3)Cl2], trans-[Pt(NH3)2NO2Cl], cis-[PtNH3PyCl2], cis-[Pt(NH3)2(NO2)2], and [Pt(NH3)3Cl]Cl exhibiting a low (if any) biological activity, either induced a decrease of the amplitude of the positive band in the CD spectrum of linear DNA, or did not affect the CD spectrum at all. The effect of these platinum compounds on the CD spectrum of the liquid crystalline microphase of DNA was either weak or absent. It is assumed that the specific biological action of platinum compounds of the cis-diamine type is determined by the polydentate binding to DNA: in addition to the cis-bidentate covalent binding of platinum to DNA nitrogen bases, a hydrogen bond formation between the DNA and cis-amino ligands occurs by means of protons at nitrogen atoms.     

Radiosensitization of human colorectal tumor cells by 4-nitro-5-sulfonatoimidazoles

Misonidazole, a clinically-effective 2-nitroimidazole hypoxic cell radiation sensitizer, and 12 4-nitro-5-sulfonatoimidazoles were tested in cultured human SW1116 colorectal adenocarcinoma cells for radiosensitizing efficiency. Octanol-water partition coefficients and HPLC capacity factors were determined for all agents as measurements of lipophilicity, and an excellent correlation was found between the two measurements. Cytotoxicity, in vitro glutathione reactivity, and one-electron reduction potential were also determined for each compound to evaluate potential utility as macromolecularly transported radiosensitizers. Ten members of the set were found to be 40 to 300 times more radiotoxic than misonidazole, but no correlation was found between their radiosensitizing efficiencies and the chemical and physical parameters.     

Mono- and polynuclear complexes of Pt(II) with polypyridyl ligands. Synthesis, spectroscopic and structural characterization and cytotoxic activity

An array of poly- and mononuclear complexes of Pt(II) with polypyridyl ligands is reported. The framework complexes [(PtCl(2))(2)(bpp)(2)(micro-PtCl(2))](H(2)O)(2) [bpp=2,3-bis(2-pyridyl)pyrazine], [PtCl(2)(micro-tptz)PtClNCPh]Cl [tptz=2,4,6-tris(2-pyridyl)-1,3,5-triazine], and mononuclear PtCl(2)(NH(2)dpt) [NH(2)dpt=4-amino-3,5-bis(2-pyridyl)-1,2,4-triazole] have been prepared and structurally characterized. Both neutral and ionic complexes are present, with bifunctional and monofunctional Pt(II) moieties, whose size and shape enable them to behave as novel scaffolds for DNA binding. Pt(II) complexes were tested for their biological activity. Cell viability assay and flow cytometric analysis demonstrated that these complexes, particularly [PtCl(2)(micro-tptz)PtClNCPh]Cl, were effective death inducers in human colon rectal carcinoma HT29 cells and their cytotoxic activity was higher than that exerted by cisplatin. Morphological analysis of treated HT29 cells, performed by fluorescence microscopy after Hoechst 33258 staining, showed the appearance of the typical features of apoptosis. Moreover, our results suggested that mitochondria are involved in apoptosis induced by Pt(II) complexes in HT29 cells as demonstrated by dissipation of mitochondrial transmembrane potential.     

Cytotoxicity, radiosensitization, and DNA interaction of platinum complexes of thiazin and xanthene dyes

Complexes of the platinum(II) tetrachlorodianion with positively charged nuclear dyes have been prepared in an effort to produce neutral molecules which could gain ready access to the nuclear DNA where the platinum(II) tetrachlorodianion could function as a radiosensitizing and a bifunctional alkylating agent. The thiazin dyes Thionin, Azure B, and Methylene Blue, the aminoxanthene dye Pyronin Y, and the thiazole dye Thioflavin have each been complexed to the platinum(II) tetrachlorodianion(PtCl4) in a ratio of 2:1(dye:PtCl4). Studies of the interaction of these complexes and of the dyes with the pBR322 plasmid superhelical DNA demonstrated that while each complex and dye readily associated with the DNA in a dose-dependent manner, only Pt(Thioflavin)2 and Thioflavin produced irreversible DNA changes (single-strand breaks). In exponentially growing EMT6 cells the cytotoxicity of these drugs was assessed in normally oxygenated and hypoxic cells at both pH 7.4 and 6.45. At concentrations ranging from 1 to 500 microM, Pt(Methylene Blue)2 was significantly more cytotoxic than the other thiazin dye complexes Pt(Thionin)2 and Pt(Azure B)2. The cytotoxicity of Pt(Thionin)2 and Pt(Methylene Blue)2 was increased in normally oxygenated and hypoxic cells at low pH. Both Pt(Pyronin Y)2 and Pt(Thioflavin)2 were more toxic than the thiazin complexes. Pt(Pyronin Y)2 was most cytotoxic to normally oxygenated cells at normal pH and hypoxic cells at low pH, while Pt(Thioflavin)2 was most cytotoxic to cells at low pH under both oxygenation conditions. In vitro studies of the radiosensitizing properties of these agents in EMT6 cells demonstrated that exposure to 100 microM for 1 h before and during irradiation (except for Pt[Thioflavin]2, which was assayed at 25 microM) resulted in enhancement rations of 2.5, 1.9, 1.5, and 1.5 for Pt(Azure B)2, Pt(Thionin)2, Pt(Pyronin Y)2, and Pt(Thioflavin)2, respectively, in hypoxic cells. In contrast, Pt(Methylene Blue)2 (and Methylene Blue) proved to be a radioprotector of normally oxygenated cells and did not sensitize hypoxic cells to the cytotoxic effects of radiation. In the FSaIIC fibrosarcoma in vivo administration of each drug at 100 mg/kg intraperitoneally (ip) 15 min prior to irradiation (except for Pt[Thioflavin]2, which was given at 1 mg/kg ip) showed that, with single radiation fractions of 10 and 20 Gy, dose-modifying factors of 2.1, 1.8, 1.5, and 1.2 were produced by Pt(Azure B)2, Pt(Thionin)2, Pt(Pyronin Y), and Pt(Methylene Blue)2, respectively, after correcting for growth delays induced by the drug alone. In comparison, misonidazole at 1 g/kg ip produced a dose-modifying factor of 1.4.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reactions of cisplatin hydrolytes with methionine, cysteine, and plasma ultrafiltrate studied by a combination of HPLC and NMR techniques

The reactions of cis-[PtCl(NH3)2(H2O)]+ with L-methionine have been studied by 1D 195Pt and 15N NMR, and by 2D[1H, 15N] NMR. When the platinum complex is in excess, the initial product, cis-[PtCl(NH3)2(Hmet-S)]+ undergoes slow ring closure to [Pt(NH3)2(Hmet-N,S)]2+. Slow ammine loss then occurs to give the isomer of [PtCl(NH3)(Hmet-N,S)]+ with chloride trans to sulfur. When methionine is in excess, a reaction sequence is proposed in which trans-[PtCl(NH3)(Hmet-S)2]+ isomerises to the cis-isomer, with subsequent ring closure reactions leading to cis-[Pt(Hmet-N,S)2]2+. Near pH 7, methionine is unreactive toward cis-[PtCl(OH)(NH3)2]. By contrast, L-cysteine reacts readily with cis-[PtCl(OH)(NH3)2] at pH 7, but there were many reaction products, including bridged species. Cis-[PtCl(OH)(NH3)2] reacts with reduced thiols in ultrafiltered plasma but these are oxidized if the plasma is not fresh or appropriately stored. With very low concentrations of the platinum complexes (35.5 microM), HPLC experiments (UV detection at 305 nm) indicate that the thiolate (probably cysteine) reactions become simpler as bridging becomes less important.     

Conformation, protein recognition and repair of DNA interstrand and intrastrand cross-links of antitumor trans-[PtCl2(NH3)(thiazole)

Replacement of one ammine in clinically ineffective trans-[PtCl2(NH3)2] (transplatin) by a planar N-heterocycle, thiazole, results in significantly enhanced cytotoxicity. Unlike 'classical' cisplatin {cis-[PtCl2(NH3)2]} or transplatin, modification of DNA by this prototypical cytotoxic transplatinum complex trans-[PtCl2(NH3)(thiazole)] (trans-PtTz) leads to monofunctional and bifunctional intra or interstrand adducts in roughly equal proportions. DNA fragments containing site-specific bifunctional DNA adducts of trans-PtTz were prepared. The structural distortions induced in DNA by these adducts and their consequences for high-mobility group protein recognition, DNA polymerization and nucleotide excision repair were assessed in cell-free media by biochemical methods. Whereas monofunctional adducts of trans-PtTz behave similar to the major intrastrand adduct of cisplatin [J. Kasparkova, O. Novakova, N. Farrell and V. Brabec (2003) Biochemistry, 42, 792-800], bifunctional cross-links behave distinctly differently. The results suggest that the multiple DNA lesions available to trans-planaramine complexes may all contribute substantially to their cytotoxicity so that the overall drug cytotoxicity could be the sum of the contributions of each of these adducts. However, acquisition of drug resistance could be a relatively rare event, since it would have to entail resistance to or tolerance of multiple, structurally dissimilar DNA lesions.     

Influence of the position of substituents in the cytotoxic activity of trans platinum complexes with hydroxymethyl pyridines

The synthesis and chemical characterization of two trans platinum complexes, (1) trans-[PtCl(2)NH(3)(2-hydroxymethylpyridine)] and (2) trans-[PtCl(2)NH(3)(3-hydroxymethylpyridine)], are described. The structures and chemical behaviour of these compounds have been compared to those of their isomer (3) trans-[PtCl(2)NH(3)(4-hydroxymethylpyridine)] previously studied. X-ray structures of all of them were solved and some interesting differences were found. The values of the dihedral angle (85 degrees , 57 degrees and 42 degrees for 1, 2 and 3, respectively) demonstrate how important is the position of substituent from a structural point of view. Studies of circular dichroism (CD), electrophoretic mobility (EM) in agarose gel and atomic force microscopy (AFM) showed differences in the modifications caused by the three complexes on DNA. Studies of antiproliferative activity of complexes 1 and 2 against cell tumour lines (HL-60) and apoptosis assays have also been carried out, showing that 1 as well as 2 are far less active than the previously described complex 3 (IC(50)=19; 19 and 3 microM, respectively). This fact probes that slight modifications on the drug's design may generate significant differences in the final antitumour activity by modifying the DNA-drug adducts, performance of resistance mechanisms and all the factors that play a fundamental role in Pt complexes' cytotoxicity.     

Activation of trans geometry in bifunctional mononuclear platinum complexes by a piperidine ligand. Mechanistic studies on antitumor action

A paradigm for the structure-pharmacological activity relationship of bifunctional platinum antitumor drugs is that the trans isomer of antitumor cisplatin (transplatin) is clinically ineffective. To this end, however, several new complexes of the trans structure have been identified that exhibit cytotoxicity in tumor cells that is even better than that of the analogous cis isomers. We reported recently (Kasparkova, J., Marini, V., Najajreh, Y., Gibson, D., and Brabec, V. (2003) Biochemistry 42, 6321-6332) that the replacement of one ammine ligand by the heterocyclic ligand, such as piperidine, piperazine, or 4-picoline in the molecule of transplatin resulted in a radical enhancement of its cytotoxicity. We examined oligodeoxyribonucleotide duplexes bearing a site-specific cross-link of the transplatin analogue containing the piperidine ligand by biochemical methods. The results indicate that in contrast to transplatin, trans-(PtCl2(NH3)(piperidine)) forms stable 1,3-intrastrand cross-links in double-helical DNA that distort DNA and are not readily removed from DNA by nucleotide excision repair system. Hence, the intrastrand cross-links of trans-(PtCl2(NH3)(piperidine)) could persist for a sufficiently long time, potentiating its toxicity toward tumor cells. trans-(PtCl2(NH3)(piperidine)) also forms in DNA minor interstrand cross-links that are similar to those of transplatin so that these adducts appear less likely candidates for genotoxic lesion responsible for antitumor effects of trans-(PtCl2(NH3)(piperidine)). Hence, the role of structurally unique intrastrand cross-links in the anti-tumor effects of transplatin analogues in which one ammine group is replaced by a heterocyclic ligand may predominate.     

Preparation, cytotoxicity and interactions with nucleophiles of three isomeric transplatinum complexes containing methylpiperidine ligands

Three isomeric complexes, trans-[PtCl2(NH3)(2-methylpiperidine)], trans-[PtCl2(NH3)(3-methylpiperidine)] and trans-[PtCl2(NH3)(4-methylpiperidine)], were prepared and their cytotoxicities against six ovarian cancer cell lines, three sensitive and three resistant to cisplatin, were measured. There were no significant differences in the cytotoxicities of the three isomers against these cell lines. The interactions of the three complexes with reduced glutathione (GSH) and with ubiquitin (Ub), as a model protein, were studied. The trans-[PtCl2(NH3)(2-methylpiperidine)] reacted approximately twice as slowly with GSH as did the other two isomers. In the 1:1 interactions of the three complexes with ubiquitin (Mr = 8565 amu), trans-[PtCl2(NH3)(3-methylpiperidine)] and trans-[PtCl2(NH3)(4-methylpiperidine)] attained 100% modification while trans-[PtCl2(NH3)(2-methylpiperidine)] reached only less than 50% modification. Trans-[PtCl2(NH3)(2-methylpiperidine)] reacts significantly less efficiently with GSH and proteins than the other two isomers yet this is not reflected in the cytotoxicity values. These results indicate that for these complexes, in these cell lines, cytosolic detoxification probably does not play a dominant role in determining the cytotoxicity of the complexes.     

Optical properties of DNA complexes with antitumor compounds of bivalent platinum

The optical properties of the DNA complexes with the compounds of bivalent platinum were studied. The compounds differed by the nature of the anionic and neutral ligands and their spatial arrangement about the platinum atom. It was shown that the same as cis-[Pt (NH3)2Cl2] the platinum compounds with the biological activity, i.e. [Pt (en) Cl2], cis-[PtNH3 (Bz) Cl2] and cis-[Pt (NH3)2NO2Cl] induced at low values of r (a ratio of the number of the platinum moles added to the number of the DNA nucleotide moles in the solution) an increase in the amplitude of the positive band in the spectrum of the circular dichroism (CD) of the linear DNA and a marked decrease in the amplitude of the negative band in the spectrum of the CD of the liquid crystalline microphase of DNA formed in the presence of polyethyleneglycol. By the character of the action on the CD spectrum of the linear and condensed DNA [Pt (tetrameen)Cl2] which had no selective antimitotic effect might be referred to the above platinum compounds. Trans-[Pt (NH3)2NO2Cl], [PtNH3PyCl2], cis-[Pt (NH3)2(NO2)2] and [Pt (NH3)3Cl]Cl having no biological activity either induced only a decrease in the amplitude of the positive band in the CD spectrum of the linear DNA or had no effect on the CD spectrum. The effect of these compounds on the CD spectrum of the liquid crystalline microphase of DNA was slightly pronounced or not observed.