A cyclopalladated complex interacts with mitochondrial membrane thiol-groups and induces the apoptotic intrinsic pathway in murine and cisplatin-resistant human tumor cells

<p>Abstract</p> <p>Background</p> <p>Systemic therapy for cancer metastatic lesions is difficult and generally renders a poor clinical response. Structural analogs of cisplatin, the most widely used synthetic metal complexes, show toxic side-effects and tumor cell resistance. Recently, palladium complexes with increased stability are being investigated to circumvent these limitations, and a biphosphinic cyclopalladated complex {Pd₂[<it>S_(-)</it>C², N-dmpa]₂(μ-dppe)Cl₂} named C7a efficiently controls the subcutaneous development of B16F10-Nex2 murine melanoma in syngeneic mice. Presently, we investigated the melanoma cell killing mechanism induced by C7a, and extended preclinical studies.</p> <p>Methods</p> <p>B16F10-Nex2 cells were treated <it>in vitro </it>with C7a in the presence/absence of DTT, and several parameters related to apoptosis induction were evaluated. Preclinical studies were performed, and mice were endovenously inoculated with B16F10-Nex2 cells, intraperitoneally treated with C7a, and lung metastatic nodules were counted. The cytotoxic effects and the respiratory metabolism were also determined in human tumor cell lines treated <it>in vitro </it>with C7a.</p> <p>Results</p> <p>Cyclopalladated complex interacts with thiol groups on the mitochondrial membrane proteins, causes dissipation of the mitochondrial membrane potential, and induces Bax translocation from the cytosol to mitochondria, colocalizing with a mitochondrial tracker. C7a also induced an increase in cytosolic calcium concentration, mainly from intracellular compartments, and a significant decrease in the ATP levels. Activation of effector caspases, chromatin condensation and DNA degradation, suggested that C7a activates the apoptotic intrinsic pathway in murine melanoma cells. In the preclinical studies, the C7a complex protected against murine metastatic melanoma and induced death in several human tumor cell lineages <it>in vitro</it>, including cisplatin-resistant ones. The mitochondria-dependent cell death was also induced by C7a in human tumor cells.</p> <p>Conclusions</p> <p>The cyclopalladated C7a complex is an effective chemotherapeutic anticancer compound against primary and metastatic murine and human tumors, including cisplatin-resistant cells, inducing apoptotic cell death via the intrinsic pathway.</p

Genus Paracoccidioides: Species Recognition and Biogeographic Aspects

Background: Paracoccidioidomycosis is a systemic mycosis caused by Paracoccidioides brasiliensis (species S1, PS2, PS3), and Paracoccidioides lutzii. This work aimed to differentiate species within the genus Paracoccidioides, without applying multilocus sequencing, as well as to obtain knowledge of the possible speciation processes. Methodology/Principal Findings: Single nucleotide polymorphism analysis on GP43, ARF and PRP8 intein genes successfully distinguished isolates into four different species. Morphological evaluation indicated that elongated conidia were observed exclusively in P. lutzii isolates, while all other species (S1, PS2 and PS3) were indistinguishable. To evaluate the biogeographic events that led to the current geographic distribution of Paracoccidioides species and their sister species, Nested Clade and Likelihood Analysis of Geographic Range Evolution (LAGRANGE) analyses were applied. The radiation of Paracoccidioides started in northwest South America, around 11–32 million years ago, as calculated on the basis of ARF substitution rate, in the BEAST program. Vicariance was responsible for the divergence among S1, PS2 and P. lutzii and a recent dispersal generated the PS3 species, restricted to Colombia. Taking into account the ancestral areas revealed by the LAGRANGE analysis and the major geographic distribution of L. loboi in the Amazon basin, a region strongly affected by the Andes uplift and marine incursions in the Cenozoic era, we also speculate about the effect of these geological events on the vicariance between Paracoccidioides and L. loboi. Conclusions/Significance: The use of at least 3 SNPs, but not morphological criteria, as markers allows us to distinguish among the four cryptic species of the genus Paracoccidioides. The work also presents a biogeographic study speculating on how these species might have diverged in South America, thus contributing to elucidating evolutionary aspects of the genus Paracoccidioides

Virulence profiles of ten Paracoccidioides brasiliensis isolates obtained from armadillos (Dasypus novemcinctus)

Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis ( PCM), the most important systemic mycosis in Latin America. The armadillo, Dasypus novemcinctus, has been confirmed as the primary natural reservoir of this fungus. Its geographic distribution is similar to that of human PCM. In this study, virulence profiles of 10 P. brasiliensis isolates from different armadillos and of two clinical isolates were tested in an experimental hamster model. Pathogenicity was evaluated by counting cfu and performing histopathological analysis in the testis, liver, spleen and lung. Circulating specific antibodies were measured using enzyme- linked immunosorbent assay ( ELISA). All isolates from armadillos were virulent in the model, with dissemination to many organs. The clinical isolates, which had long been stored in cultured collections, were less virulent. The isolates were classified into four virulence categories according to number of cfu per gram of tissue: very high, high, intermediate and low. This study confirms that armadillos harbor pathogenic genotypes of P. brasiliensis, probably the same ones that infect humans

Virulence attenuation and phenotypic variation of Paracoccidioides brasiliensis isolates obtained from armadillos and patients

Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, the most important systemic mycosis in Latin America. The virulence profiles of five isolates of P. brasiliensis were studied in two different moments and correlated with some colonial phenotypic aspects. We observed a significant decrease in the virulence and an intense phenotypic variation in the mycelial colony. The recognition of all ranges of phenotypic and virulence variation of P. brasiliensis, as well as its physiological and genetic basis, will be important for a better comprehension of its pathogenic and epidemiological features

High frequency of Paracoccidioides brasiliensis infection in armadillos (Dasypus novemcinctus): an ecological study

The fungus Paracoccidioides brasiliensis has been isolated from nine-banded armadillos (Dasypus novemcinctus) in different regions where paracoccidiodomycosis (PCM) is endemic. the link between PCM and these animals has provided the first valuable clue in the effort to elucidate the ecological niche of P. brasiliensis. the present study was aimed at correlating P. brasiliensis infection in armadillos with local ecological features and, if possible, the presence of the fungus in the soil in the Botucatu hyperendemic area of PCM. in this region the mean temperature ranges from 14.8 to 25.8degreesC and the annual average precipitation is 1520 mm. the sites where 10 infected animals (positive group) were collected were studied and compared with the sites where five uninfected animals were found. the occurrence of the fungus in soil samples collected from the positive armadillos' burrows and foraging sites was investigated by the indirect method of animal inoculation. Environmental data from the sites of animal capture, such as temperature, rainfall, altitude, vegetation, soil composition, presence of water and proximity of urban areas, were recorded. All 37 soil samples collected from the sites had negative fungal cultures. Positive animals were found much more frequently in sites with disturbed vegetation, such as riparian forests and artificial Eucalyptus Or Pinus forests, in altitudes below 800 m, near water sources. the soil type of the sites of positive animals was mainly sandy, with medium to low concentrations of organic matter. the pH was mainly acidic at all the sites, although the concentrations of aluminum cations (H+Al) were lower at the sites where positive animals were found. Positive armadillos were also captured in sites very close to urban areas. Our data and previous studies indicate that P. brasiliensis occurs preferentially in humid and shady disturbed forests in a strong association with armadillos.Univ Estadual Paulista Julio Mesquita Filho, Inst Biociencias, Dept Microbiol & Imunol, Botucatu, SP, BrazilUNIFESP, Escola Paulista Med, Dept Patol, São Paulo, BrazilUniv Estadual Paulista Julio Mesquita Filho, Inst Biociencias, Dept Bioestatist, Botucatu, SP, BrazilUniv Estadual Paulista Julio Mesquita Filho, Fac Med, Dept Patol, Botucatu, SP, BrazilUNIFESP, Escola Paulista Med, Dept Patol, São Paulo, BrazilWeb of Scienc

The tricyclic antidepressants amitriptyline, nortriptyline and imipramine are weak antagonists of human and rat alpha(1B)-adrenoceptors

Although it is long known that the tricyclic antidepressants amitriptyline, nortriptyline and imipramine inhibit the noradrenaline transporter and alpha(1)-adrenoceptors with similar affinities, which may lead to self-cancelling actions, the selectivity of these drugs for alpha(1)-adrenoceptor subtypes is unknown. The present study investigates the selectivity of amitriptyline, nortriptyline and imipramine for human recombinant and rat native alpha(1)-adrenoceptor subtypes. The selectivity of amitriptyline, nortriptyline and imipramine was investigated in HEK-293 cells expressing each of the human alpha(1)-subtypes and in rat native receptors from the vas deferens (alpha(1A)), spleen (alpha(1B)) and aorta (alpha(1D)) through [H-3]prazosin binding, and noradrenaline-induced intracellular Ca2+ increases and contraction assays. Amitriptyline, nortriptyline and imipramine showed considerably higher affinities for alpha(1A)- (similar to 25- to 80-fold) and alpha(1D)-adrenoceptors (similar to 10- to 25-fold) than for alpha(1B)-adrenoceptors in both contraction and [H-3]prazosin binding assays with rat native and human receptors, respectively. In addition, amitriptyline, nortriptyline and imipramine were substantially more potent in the inhibition of noradrenaline-induced intracellular Ca2+ increases in HEK-293 cells expressing alpha(1A)- or a truncated version of alpha(1D)-adrenoceptors which traffics more efficiently towards the cell membrane than in cells expressing alpha(1B)-adrenoceptors.Amitriptyline, nortriptyline and imipramine are much weaker antagonists of rat and human alpha(1B)-adrenoceptors than of alpha(1A)- and alpha(1D)-adrenoceptors. The differential affinities for these receptors indicate that the alpha(1)-adrenoceptor subtype which activation is most increased by the augmented noradrenaline availability resultant from the blockade of neuronal reuptake is the alpha(1B)-adrenoceptor. This may be important for the behavioural effects of these drugs. (C) 2010 Elsevier Ltd. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq