Miguel A. Fuertes

Biochemical Mechanisms of Cisplatin Cytotoxicity

Since the discovery by Rosenberg and collaborators of the antitumor activity of cisplatin 35 years ago, three platinum antitumor drugs (cisplatin, carboplatin and oxaliplatin) have enjoyed a huge clinical and commercial hit. Ever since the initial discovery of the anticancer activity of cisplatin, major efforts have been devoted to elucidate the biochemical mechanisms of antitumor activity of cisplatin in order to be able to rationally design novel platinum based drugs with superior pharmacological profiles. In this report we attempt to provide a current picture of the known facts pertaining to the mechanism of action of the drug, including those involved in drug uptake, DNA damage signals transduction, and cell death through apoptosis or necrosis. A deep knowledge of the biochemical mechanisms, which are triggered in the tumor cell in response to cisplatin injury not only may lead to the design of more efficient platinum antitumor drugs but also may provide new therapeutic strategies based on the biochemical modulation of cisplatin activity.

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.

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.

Poly(ADP-Ribose) Polymerase-1 (PARP-1) Inhibitors in Cancer Chemotherapy

Poly(ADP-ribose) polymerases (PARPs) are defined as a family of cell signaling enzymes present in eukaryotes, which are involved in poly(ADP-ribosylation) of DNA-binding proteins. The best studied of these enzymes (PARP-1) is involved in the cellular response to DNA damage so that in the event of irreparable DNA damage overactivation of PARP-1 leads to necrotic cell death. Inhibitors of PARP-1 activity in combination with DNA-binding antitumor drugs may constitute a suitable strategy in cancer chemotherapy. When DNA is moderately damaged, PARP-1 participates in the DNA repair process and the cell survives. However, in the case of extensive DNA damage PARP-1 overactivation induces a decrease of NAD+ and ATP levels leading to cell dysfunction or even to necrotic cell death. So, due to PARP-1 involvement in cell death, pharmacological inhibition of PARP-1 activity by PARP-1 inhibitors may constitute a suitable target to enhance the activity of antitumor drugs through inhibition of necrosis and activation of apoptosis. PARP-1 inhibitors such as 3-aminobenzamide, 1,5-dihydroxyisoquinolinone and the recently patented tryciclic benzimidazoles have shown potent inhibitory effects of PARP-1 activity in tumor cells. The present review gives an update of the state-of-the-art of inhibition of PARP-1 activity as adjuvant therapy in cancer treatment.

Poly(ADP-ribose) Polymerase-1 Inhibitor 3-Aminobenzamide Enhances Apoptosis Induction by Platinum Complexes in Cisplatin-Resistant Tumor Cells

Cisplatin is one of the most widely used antitumor drugs. However, as all the anticancer drugs currently used in clinic, cisplatin shows the phenomenon of drug resistance (intrinsic or acquired) against a wide variety of tumors. Poly (ADP-ribose) polymerase-1 is an enzyme involved in DNA repair and apoptotic cell death, which may be inhibited to increase cisplatin chemosensitivity of tumor cells so that cisplatin resistance may be circumvented. In the present study we report that PARP-1 inhibitor 3-aminobenzamide (3-AB) increases the cytotoxic activity of the platinum compounds cisplatin, trans-[PtCl(2)(4-picoline)(piperazine)] and transplatin against CH1cisR cisplatin-resistant ovarian tumor cells. In fact, a concentration of 3-AB of 1 mM not only increases the cytotoxic activity of these platinum complexes but also switches the mode of cell death from necrosis to apoptosis. Altogether, these data suggest that pharmacological modulation of PARP-1 by inhibitors may be a suitable strategy to fight against tumor resistance to platinum drugs.

Anticancer compounds as leishmanicidal drugs: challenges in chemotherapy and future perspectives.

Leishmaniasis comprises a spectrum of parasitic illnesses caused by several species of the protozoan kinetoplastid parasite, Leishmania spp. The disease affects 12 million people around the world with an annual death rate of approximately 80,000 people. Several drugs are available for treating leishmaniasis. For example, pentavalent antimonial compounds, such as sodium stibogluconate and meglumine antimonite are the drugs used in first-line chemotherapy. As second-line drugs, amphotericin B and pentamidine are used. However, current treatments against leishmaniasis are usually unsatisfactory due to some limitations including the route of administration of the drugs, their unaffordable cost and toxicity. Efforts have been made to develop new leishmanicidal drugs and to find new strategies of drug design. Hence, it is interesting to point out that the effectiveness of certain molecules as both anticancer drugs and antiprotozoal agents suggested that this class of compounds and their derivatives might be useful as antileishmanial agents. This review summarizes the anticancer compounds that have been investigated against leishmaniasis. Some of such agents include: compounds with in vitro antileishmanial activities, molecules tested in clinical trials and registered patents. We finally discuss challenges in chemotherapy and future prospects in the treatment of leishmaniasis.

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.

Molecular recognition in the human immunodeficiency virus capsid and antiviral design

Many compounds able to interfere with HIV-1 infection have been identified; some 25 of them have been approved for clinical use. Current anti-HIV-1 therapy involves the use of drug cocktails, which reduces the probability of virus escape. However, many issues remain, including drug toxicity and the emergence of drug-resistant mutant viruses, even in treated patients. Therefore, there is a constant need for the development of new anti-HIV-1 agents targeting other molecules in the viral cycle. The capsid protein CA plays a key role in many molecular recognition events during HIV-1 morphogenesis and uncoating, and is eliciting increased interest as a promising target for antiviral intervention. This article provides a structure-based, integrated review on the CA-binding small molecules and peptides identified to date, and their effects on virus capsid assembly and stability, with emphasis on recent results not previously reviewed. As a complement, we present novel experimental results on the development and proof-of-concept application of a combinatorial approach to study molecular recognition in CA and its inhibition by peptide compounds.

Apoptosis induction and DNA interstrand cross-link formation by cytotoxic trans-[PtCl2(NH(CH3)2)(NHCH(CH3)2)]: Cross-linking between d(G) and complementary d(C) within oligonucleotide duplexes

We have investigated the cytotoxic activity, the induction of apoptosis, and the interstrand cross‐linking efficiency in the A2780cisR ovarian tumor cell line, after replacement of the two NH3 nonleaving groups in trans‐[PtCl2(NH3)2] (trans‐DDP) by dimethylamine and isopropylamine. The data show that trans‐[PtCl2(NH(CH3)2)(NHCH(CH3)2)] is able to circumvent resistance to cis‐[PtCl2(NH3)2] (cis‐DDP, cisplatin) in A2780cisR cells. In fact, trans‐[PtCl2(NH(CH3)2)(NHCH(CH3)2)] shows a cytotoxic potency higher than that of cis‐DDP and trans‐DDP, with the mean IC50 values being 11, 58, and 300 μM, respectively. In addition, at equitoxic doses (concentrations of the platinum drugs equal to their IC50 values) and after 24 hours of drug treatment, the level of induction of apoptosis by trans‐[PtCl2(NH(CH3)2)(NHCH(CH3)2)] is twice that produced by cis‐DDP. Under the same experimental conditions, trans‐DDP does not induce significant levels of apoptosis in A2780cisR cells. After 24 hours of incubation of A2780cisR cells at concentrations equal to the IC50 value of the platinum drugs, the level of DNA interstrand cross‐links (ICLs) induced by trans‐[PtCl2(NH(CH3)2)(NHCH(CH3)2)] is two and three times higher, respectively, than those induced by cis‐DDP and trans‐DDP. We also found that trans‐[PtCl2(NH(CH3)2)(NHCH(CH3)2)] formed DNA ICLs between guanine and complementary cytosine. We propose that, in A2780cisR cells, the induction of apoptosis by trans‐[PtCl2(NH(CH3)2)(NHCH(CH3)2)] is related to its greater ability (relative to cis‐DDP and trans‐DDP) to form DNA ICLs.

Neospora caninum infection as a cause of reproductive failure in a sheep flock

Neospora caninum has been detected only sporadically in cases of ovine abortion, and it has therefore traditionally been considered as an unimportant parasite in small ruminants. This study was carried out with the aim of identifying the pathogen causing serious reproductive problems on a commercial sheep farm. Sera from all rams and ewes tested negative for antibodies against Border disease virus, Schmallenberg virus and Coxiella burnetii, and infections by these agents were therefore ruled out. Nevertheless, seropositivity to N. caninum and/or Toxoplasma gondii was detected, although the seroprevalence was higher in the case of N. caninum. The percentage of lambings and the number of lambs per dam were significantly lower in ewes that were seropositive to N. caninum while no effect on these parameters was detected in ewes that were seropositive to T. gondii. There was also no evidence of infection by T. gondii in the foetal/lamb tissues analyzed by PCR and/or immunohistopathological techniques. On the contrary, the DNA of N. caninum was detected in 13 out of 14 foetuses/lambs descendant from dams seropositive to this parasite. Characteristic lesions caused by N. caninum and/or its antigen were also detected. Genotyping of the N. caninum DNA revealed only two closely related microsatellite multilocus genotypes. The results clearly demonstrate that infection by N. caninum was the cause of the low reproductive performance of this sheep flock.