Current status of the development of trans-platinum antitumor drugs.

The discovery in the 1990s of several trans-Pt complexes with in vitro and in vivo activity against tumor cells sensitive and/or resistant to cisplatin has forced the re-evaluation of the structure-activity relationships for platinum antitumor drugs. Because the determinant factors of cytotoxic activity of trans-platinum complexes do not follow the same patterns as those found for cisplatin and its analogues, the differences in cellular and biochemical pharmacology between trans-platinum antitumor complexes and cisplatin might be systematically exploited to design novel trans-platinum complexes with a clinical profile complementary to that of cisplatin and related analogues. Therefore, there may exist a novel molecular rationale for new platinum antitumor drugs development in the twenty-first century.

Cisplatin biochemical mechanism of action: from cytotoxicity to induction of cell death through interconnections between apoptotic and necrotic pathways.

Although cisplatin, cis-diamminedichloroplatinum(II), has been successfully used in the chemotherapy of cancer for more than 25 years, its biochemical mechanism of action is still unclear. The current accepted paradigm about cisplatin mechanism of action is that the drug induces its cytotoxic properties through binding to nuclear DNA and subsequent interference with normal transcription, and/or DNA replication mechanisms. If cisplatin-DNA adducts are not efficiently processed by cell machinery, cytotoxic processes eventually end up in cell death. However, before cisplatin enters the cell it may bind to phospholipids and phosphatidylserine in the cell membrane. In addition, in the cytoplasm many potential platinum-binding sites are also available, including RNA and sulfur-containing biomolecules. Moreover, there is much evidence suggesting that the cytotoxic effects induced by binding of cisplatin to non-DNA targets (especially proteins) may contribute to its biochemical mechanism of action. On the other hand, it has been found that several factors such as the dose of drug as well as the metabolic condition of the cell subjected to cisplatin aggression, may determine that cancer cells die through apoptosis or necrosis. In fact, it has recently been reported that both mechanisms of cell demise work in concert so that within a population of tumour cells there is a continuum of possible modes of cell death.

Novel concepts in the development of platinum antitumor drugs.

The limitations of cisplatin as an anticancer drug have stimulated the search for other antitumor-active platinum complexes with improved pharmacological properties. The two main goals in the search for new platinum anti-cancer agents are the reduction of the dose-limiting toxicities of cisplatin and the circumvention of cisplatin resistance. However, it should be pointed out that this has proven to be a difficult task. In fact, less than 1% of the thousand of platinum complexes tested for pre-clinical antitumor activity have entered clinical trials in the past 30 years. Nonetheless, right now, several new platinum complexes are in clinical trials, a proof of the continued belief that platinum complexes may still fulfil the needs for novel antitumor drugs. This review will focus on the three main innovative approaches found in the platinum anticancer-field, namely, (1) compounds with decreased reactivity against nucleophiles, (2) compounds with carrier ligands, and (3) compounds which bind differently to DNA as compared to cisplatin. In the latter class, special attention is paid to dinuclear and polinuclear platinum complexes.

X-Ray structure of cytotoxic trans-[PtCl(2)(dimethylamine)(isopropylamine)]: interstrand cross-link efficiency, DNA sequence specificity, and inhibition of the B-Z transition.

We report here the X-ray structure of cytotoxic trans-¿PtCl(2)(dimethylamine)(isopropylamine). This trans-platinum compound crystallizes in the monoclinic system, with Z = 8, in the spatial group C2/c with unit cell parameters a = 19.862(17) A, b = 6. 581(3) A, c = 18.563(3) A, alpha = 90 degrees, beta = 119.16(3) degrees, gamma = 90 degrees, V = 2119(2) A(3), rho = 2.321 Mg/m(3), R = 0.0505, and R(w) = 0.1166 on the basis of 2339 independent reflections. To our knowledge this is the first report of the crystal structure of a biologically active trans-platinum compound containing different aliphatic amines. The DNA binding mode of trans-¿PtCl(2)(dimethylamine)(isopropylamine) may be a consequence of the spatial disposition of the dimethylamine and isopropylamine ligands around the trans-Pt(II) center. We have found that trans-¿PtCl(2)(dimethylamine)(isopropylamine) readily forms DNA interstrand cross-links. In addition, the compound shows binding affinity toward alternating purine-pyrimidine sequences and inhibits the B-Z transition. These particular DNA binding properties might be related to the capacity of trans-¿PtCl(2)(dimethylamine)(isopropylamine) for inducing some selective killing in a H-ras overexpresssing cell line.

Modification of the recognition of restriction sites of plasmid DNA by the antitumor drug Pt-pentamidine.

The rate of binding of the antineoplastic drugs Pt-pentamidine [(cis-PtCl2)3(pentamidine)3][PtCl4]2 and cis-DDP [cis-diamminedichloroplatimum(II)] to pUC8 DNA, as well as the effect of the binding of these platinum compounds on the cutting effectiveness of Bam HI, Hind III, and Sal I restriction endonucleases, were determined by flameless atomic absorption spectroscopy and gel electrophoresis, respectively. The results show that covalent DNA platination is 12% to 22% lower in DNA: Pt-pentamidine complexes than in DNA: cis-DDP at the same molar rate of platinum/nucleotide, and the number of Pt-pentamidine molecules bound to DNA is significantly lower in Pt-pentamidine: DNA complexes than in cis-DDP: DNA complexes. Although both compounds inhibit Bam HI cleavage of pUC8 DNA, Pt-pentamidine does not prevent the cutting activity of Hind III, in contrast with cis-DDP. Neither cis-DDP nor Pt-pentamidine inhibits the cutting activity of Sal I, whose recognition sequence neighbors the Bam HI and Hind III sites.

Induction of apoptosis by the bis-Pt(III) complex [Pt(2)(2-mercaptopyrimidine)(4)Cl(2)].

We analyzed both the cytotoxicity and the type of cell death produced by the novel binuclear Pt(III) compound Pt-Spym ([Pt(2)(2-mercaptopyrimidine)(4)Cl(2)]) in kidney human fibroblasts and in human tumor cell lines (HeLa, CH1, CH1cisR and HL-60). The data showed that Pt-Spym displayed higher cytotoxicity against these tumor cells than cisplatin. In contrast, Pt-Spym had low toxicity against normal human fibroblasts. Interestingly, Pt-Spym circumvented cisplatin resistance in CH1cisR cells. We also observed that Pt-Spym induced the characteristic changes attributed to apoptosis in cells with normal levels of p53 protein (CH1 and CH1cisR) and with low levels of p53 protein (HeLa), but not in cells lacking p53 (HL-60). Interestingly, Western blot data indicated that apoptosis induction by Pt-Spym in HeLa, CH1, and CH1cisR cells was not associated with drastic changes in p53 levels. However, cis-DDP strongly decreased p53 levels in CH1 and CH1cisR cells and abolish p53 protein in HeLa cells. Altogether, these results suggest that induction of apoptosis by Pt-Spym requires the presence of p53 protein.

Synthesis, characterization, and DNA modification induced by a novel Pt-berenil compound with cytotoxic activity.

In the present paper, we show that the reaction of the antipyranosomatid berenil drug with K2PtCl4 resulted in the synthesis of a covalent (Pt(II)-berenil compound of formula [Pt2Cl4(berenil)2]Cl4.4H2O as shown by IR, 1H, 13C, and 195Pt-NMR. The Pt-berenil compound was tested for in vitro antitumor activity against HL-60 and U-937 human leukemic cells. The results show that the LC70 values of the Pt-berenil are about two-fold lower than those of cis-DDP in both HL-60 and U-937 cell lines. Melting data of Pt-berenil:DNA and berenil:DNA complexes indicate that the platinated compound produces on a DNA secondary structure higher compaction than the berenil ligand. The mobility in agarose gels and the circular dichroism spectra of the compounds:DNA complexes revealed, moreover, that both induce drastic changes on a DNA secondary and tertiary structure. The total reflection X-ray fluorescence data showed, in additIon that DNA platination in Pt-berenil:DNA complexes occurs within minutes after addition of the drug, in contrast to what that observed in cis-DDP:DNA complexes. On the basis of these results, we propose that in Pt-berenil, the berenil ligand acts as a carrier of the active cis-P(II) centers towards DNA.

Application of a mathematical model to describe the behaviour of the Lactobacillus spp. during the ripening of a Spanish dry fermented sausage (Chorizo).

The evolution of Lactobacillus spp. during the curing process of dry fermented sausage (Chorizo), has been studied under natural climatic conditions and in a controlled drying chamber, with regulated temperature and relative humidity. In order to fully understand the development of the microorganisms in each of these two cases, it applied a mathematical model based on a modified Gompertz equation. The proposed mathematical function is easy to use and statistically appropriate. Furthermore, it allows the distinction to be made between the four phases which characterise the behaviour of these microorganisms during the curing of the sausage (a) latency from an initial threshold level; (b) exponential growth; (c) stationary state; (d) depletion to a residual level. The mathematical analysis of this function has highlighted certain differences in the behaviour of the microbial flora depending on the technological conditions to which the drying of the sausages is subjected. Under the climatically controlled conditions of the drying process, a better development of the lactobacillus spp. is observed, but conversely the final stage of the exponential growth is brought forward, the length of the stationary phase is shortened and is reached before the end of the drying period, at which point the marginal decline of the microorganisms becomes ever more reduced.

UV-B absorbing compounds in present-day and fossil pollen, spores, cuticles, seed coats and wood: evaluation of a proxy for solar UV radiation.

UV-B absorbing compounds (UACs) in present-day and fossil pollen, spores, cuticles, seed coats and wood have been evaluated as a proxy for past UV. This proxy may not only provide information on variation of stratospheric ozone and solar UV in the period preceding and during the Antarctic ozone hole (1974-present day), but also on the development and variation of the stratospheric ozone layer and solar surface UV during the evolution of life on Earth. Sporopollenin and cutin are highly resistant biopolymers, preserving well in the geological record and contain the phenolic acids p-coumaric (pCA) and ferulic acid (FA). pCA and FA represent a good perspective for a plant-based proxy for past surface UV radiation since they are induced by solar UV-B via the phenylpropanoid pathway (PPP). UV-B absorption by these monomers in the wall of pollen and spores and in cuticles may prevent damage to the cellular metabolism. Increased pCA and FA in pollen of Vicia faba exposed to enhanced UV-B was found in greenhouse experiments. Further correlative evidence comes from UV-absorbing compounds in spores from 1960-2000 comparing exposure of land plants (Lycopodium species) to solar UV before and during ozone depletion and comparing plants from Antarctica (severe ozone depletion), Arctic, and other latitudes with less or negligible ozone depletion. Wood-derived compounds guaiacyl (G), syringyl (S), and p-hydroxyphenyl (P) are produced via the PPP. The proportions of P, G, and S in the lignin differ between various plant groups (e.g. dicotyledons/monocotyledons, gymnosperms/angiosperms). It is hypothesized that this lignin composition and derived physiological and physical properties of lignin (such as tree-ring wood density) has potential as a proxy for palaeo-UV climate. However validation by exposure of trees to enhanced UV is lacking. pCA and FA also form part of cutin polymers and are found in extant and fossil Ginkgo leaf cuticles as shown by thermally-assisted hydrolysis and methylation (THM)-pyrolysis-GC-MS. Potentially, the time scale for reconstruction of ozone column thickness and UV-B based on the UAC UV proxy may be decadal, centennial, millennial and possibly billenial. For further development of the UACs and pCA and FA-based UV proxy, it is necessary to obtain the UV dose-response (content of UACs, pCA and FA in sporopollenin and cutin) relationships for validation, based on outdoor UV radiation manipulations experiments with plants, and comparative analysis of stored plants (herbaria) or fossil material of the same or related plant species.

Application of a Mathematical Model for the Inhibition of Enterobacteriaceae and Clostridia during a Sausage Curing Process.

The application of a mathematical model capable of providing expressions suitable for analysis of the population changes of Enterobacteriaceae and clostridia during the curing process of dry fermented sausage is proposed to facilitate better control of the safety of the product. The mathematical model provides curing times that are crucial for control of the safety of the product: the beginning of the exponential phase of inhibition, the time during which the rate of inhibition is at a maximum, and the phase in which the rate of decline of the population of microorganisms decreases until they virtually disappear from the sausage. For Enterobacteriaceae , growth inhibition and eventual disappearance are dependent on conditions during sausage drying; for clostridia the means of drying is not significant. Under programmed controlled drying conditions, the maximum speed of inhibition and the disappearance of undesirable microorganisms are reached sooner than under natural climatic drying conditions. A regression equation has been established that relates the cell number of Enterobacteriaceae to pH and aw, values, and the cell number of Lactobacillaceae , whose metabolic activities give rise to lactic and acetic acids. The significance of the drying conditions may verify the values reached by all these parameters during curing.

Empirical validation of a two-state kinetic model for the B-Z transition of double-stranded poly[d(G-m5C)].

A formula based on a first-order kinetic equation is derived to evaluate the rate constant of the B-Z transition of a synthetic double-stranded poly[d(G-m5C)] in terms of the salt concentration, the absolute temperature, and the cooperativity index. The validity of the formula was tested using circular dichroism spectroscopy after variation of the type of salt (NaCl, MgCl2), the salt concentration, and the temperature of the polynucleotide solution. A consequence of the proposed function is that in conditions of high salt there is a predictable salt threshold which determines the particular molecular mechanism of the B-Z transition. The paper also describes the way in which this threshold level is temperature dependent. A detailed comparison of our data with the experimental data found by other authors is given. The function agrees quantitatively with the experiments and explains the contrasting results found in the literature about the influence in the B-Z transition of both the temperature and the polymer size.

Improvement of scleredema associated with IgA multiple myeloma after chemotherapy.

The presence of a monoclonal gammopathy in patients with chronic scleredema is a recently described, unusual association of unknown significance. There have been very occasional reports of multiple myeloma in such patients. We now report a patient with long-standing scleredema who developed an IgA-kappa multiple myeloma. Once chemotherapy for the myeloproliferative disorder was instituted, scleredema improved significantly, suggesting a pathogenetic link between the two conditions.

Is cisplatin-induced cell death always produced by apoptosis?

It is generally accepted that DNA damage and subsequent induction of apoptosis may be the primary cytotoxic mechanism of cisplatin and other DNA-binding antitumor drugs (Fisher,1994). Because the final step of apoptosis is characterized by morphological changes in the nucleus, the death signals of the execution phase must be transmitted from the cytoplasm to the nucleus. Thus, the recognition and processing of cisplatin-induced DNA damage through"classic" apoptosis, requires that a nuclear signal, generated at the initiation phase, be transmitted to the cytoplasm to be processed through the effector and execution phases. At the end of the execution phase, the apoptotic signal must come back to the nucleus to produce internucleosomal DNA degradation. Therefore, the induction of apoptosis from detection and subsequent processing of cisplatin-induced DNA damage seems to be a long and complex process of cell death. However, because cisplatin is a nonspecific drug and reacts not only with DNA but also with proteins,we cannot rule out the possibility that in some cases of cisplatin-induced apoptosis, an easier process of initiation, such as damage to cytoplasmic proteins, may take place (Pérez, 1998). Thus, damage to proteins is worth considering as a factor contributing to cisplatin-induced apoptosis. Moreover, it is possible that cisplatin damage to proteins could induce apoptosis at the execution phase level. In fact, initiation of apoptosis at the execution phase (activation of caspases) has been previously reported for the cell killing produced by cytotoxic T lymphocytes (Golstein et al., 1991). Although apoptosis and necrosis are conceptually distinct forms of cell death with very different morphological and biochemical characteristics, these two types of demise may occur simultaneously in tissues or cell cultures exposed to the same insult (Eguchi et al., 1997, Zhan et al., 1999). In fact, both types of cell death have been found in the same population of cisplatin-treated cells (Pestell et al., 2000). Moreover, it has been hypothesized that in a tissue or cell population,apoptosis and necrosis might be two extremes of a continuum of possible types of cell demise. Individual cell death would be decided by factors such as the availability of energy and the metabolic condition of the cell (Leist et al., 1997). Thus, some cells might die as a result of an unfinished apoptotic program. In fact, in L1210 leukemic cells, cisplatin-induced cell death seems to be the result of a defective apoptotic program that lacks some morphological and biochemical characteristics attributed to apoptosis (Segal-Bendirdjian and Jacquemin-Sablon, 1995). In addition, at high doses, cisplatin could damage molecules involved in cellular energy supply (i.e., ATP) and also proteins directly or indirectly involved in the apoptotic process (i.e., p53, Bax, Bcl-2, and caspases), leading to necrotic cell death. In fact, in cisplatin-resistant keratinocytes transformed by H-ras oncogene, a high dose of cisplatin (312 microM) induces characteristic features of necrotic cell death(Pérez et al., 1999). Thus, depending on the level of cellular damage induced by cisplatin, necrosis could take place either directly or as a consequence of an unfinished apoptotic program. In summary, a growing body of evidence suggests that cisplatin-induced cell death does not always come from "classic"apoptosis. Depending on both cisplatin dose and cellular status, cisplatin may also induced cell death by a defective apoptotic program or even by necrosis. Elucidation of the conditions under which the apoptotic program induced by cisplatin as well as other antitumor drugs is totally or partially executed may have important implications for the outcome of cancer chemotherapy.

Calcium-induced conformational changes in Leishmania infantum kinetoplastid membrane protein-11.

Total-reflection X-ray fluorescence has been used to study whether the Leishmania infantum kinetoplastid membrane protein-11 is a Ca2+-binding protein. The 108 amino acid helix-loop-helix protein has the loop region located between residues 45 and 57, having similarity to the EF-hand motifs. In particular, the sequence alignment of the putative motif revealed the existence of 67% similarity and 33% identity with the EF-hand of the plasmodia-specific 40-kDa protein from Physarum polycephalum. To address the type of conformational changes induced by Ca2+ binding, circular dichroism and fluorescence spectroscopy were used. The data showed that Ca2+ induces changes in both the secondary and tertiary structure of the protein in a temperature- and pH-dependent way. It also induces the precipitation of the protein at pH 7.5, in contrast with what occurs at pH 5.0, and the precipitation process can be reverted by addition of EGTA. At acidic pH values the complex EGTA-Ca2+ causes drastic structural changes, forcing the protein to adopt a structure close to that of a random coil. Because, at acidic pH values, protein:Ca2+:EGTA ternary complexes may be formed, the drastic change may be attributed to the presence of a high density of EGTA negative charges in the neighborhood of the alpha-helices.

A kinetic model for the B-Z transition of poly[d(G-C)].poly[d(G-C)] and poly[d(G-m5C)].poly[d(G-m5C)].

The analysis of the kinetic data of the B-Z conformational changes induced by salt in sized double-stranded poly[d(G-C)].poly[d(G-C)] and poly[d(G-m5C)].poly[d(G-m5C)] polymers indicated that there exists a salt threshold which reveals some largely, as yet, unrecognised characteristics of the transition. It was observed that there is a direct correlation between the length of the polymer and the rate of the B-Z transition when the salt concentration in the polymer solution is lower than the salt threshold. The correlation is inverse when the salt concentration is higher than the salt threshold. Thus, the molecular mechanism of the B- to Z-DNA transition varies depending on whether the salt concentration is higher or lower than the threshold. In this context, we have found that the contrasting results reported in the literature describing the rate of the B-Z transition are not contradictory but complementary. The finding of a salt threshold leads to the establishment of a relationship between the cooperativity index of the B-Z transition and the polymer chain length. That relationship is dependent on the chemical structure of the polymer but is temperature independent.

Folding stability of the kinetoplastid membrane protein-11 (KMP-11) from Leishmania infantum.

Kinetoplastid membrane protein-11 (KMP-11) is a major component of the cell surface of kinetoplastids, and acts as a potent B- and T-cell immunogen during Leishmania infection. Here we report that the Leishmania infantum KMP-11 secondary structure adopts mainly an alpha-helical conformation at pH 7.5 and that its urea- and thermally-induced unfolding constitute a fully reversible two-step process. This allows estimation of a half-denaturation temperature of approximately 65 degrees C, a delta GDH2O at 20 degrees C of approximately 14.63 kJ.mol-1, and an increment of the reaction heat of approximately 183.92 kJ.mol-1 and an entropy of approximately 543.4 J.mol-1.deg-1, respectively, for the native-denatured equilibrium of the KMP-11 in solution. We also report that the KPM-11 protein is induced to adopt a molten globule state at a pH range between pH 4 and pH 6. As a whole, the stability and the specific features of the denaturing effect induced by changes in pH are similar in KMP-11 to various other lipoproteins.