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

Caracterização molecular de isolados de Paracoccidioides brasiliensis obtidos de Dasypus novemcinctus (tatu) e sua correlação com virulência e origem geográfica

O fungo dimórfico Paracoccidioides brasiliensis (Pb) é o agente etiológico da paracoccidioidomicose (PCM), a micose sistêmica mais importante da América Latina. A espécie de tatu Dasypus novemcinctus mostrou ser um importante reservatório do fungo e o estudo de isolados destes animais será útil para uma melhor compreensão da ecologia e epidemiologia desta doença. O objetivo deste trabalho foi comparar a virulência e variabilidade genética de isolados de tatus e de pacientes para melhor compreender a importância do tatu na PCM. Os isolados animais foram obtidos da área endêmica de Botucatu, São Paulo, Brasil, com exata informação dos locais de coleta. Os isolados clínicos, exceto a cepa padrão Pb265, foram obtidos de pacientes residentes nos municípios de Pratânia, Manduri e Botucatu. A virulência foi avaliada no modelo experimental do hamster através da contagem de UFC (Unidades Formadoras de Colônias), histopatologia e dosagem de anticorpos específicos e a caracterização molecular por meio da técnica de RAPD e sequenciamento do DNA ribossômico. Todos isolados de tatus foram virulentos, disseminando-se para vários órgãos. A cepa Pb265, isolada de paciente, apresentou baixa virulência. A contagem de UFC nos diferentes órgãos permitiu uma classificação do fungo em 4 categorias: virulência muito alta, virulência alta, virulência intermediária e baixa virulência. A análise molecular mostrou grande diversidade genética entre os isolados, porém não houve separação entre os fungos isolados de pacientes e de tatu. Estes resultados indicam que tanto o homem quanto o tatu podem estar sendo infectados por patógenos com mesmo genótipo, provavelmente localizados em foco ambiental comum. Portanto, os tatus como reservatórios do P. brasiliensis na natureza, representam importantes elos na cadeia da doença...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 first natural reservoir of this fungus and its geographic distribution is similar to the distribution of human PCM. This work analysed the virulence profiles and genetic diversity of P. brasiliensis isolates from different armadillos and clinical isolates. The animal isolates were obtained from the Botucatu endemic area of PCM, with exact information of the collection sites. The clinical isolates, except Pb265, were obtained from patients that live in the same endemic area (Pratânia, Manduri and Botucatu county). Virulence was evaluated in an experimental hamster model by colony-forming units (CFU) and histopathological analysis in the testis, liver, spleen, lung and circulating specific antibodies measured by ELISA. Molecular characterisation was evaluated by RAPD methods and sequencing analyses of the internal transcribed spacer regions (ITS 1 and ITS 2) and 5.8 S ribosomal gene. All isolates from armadillos were virulent, with dissemination to many organs. The clinical isolate Pb 265 was less virulent. The isolates were classified into four categories according to number of CFU per gram of tissue: very high, high, intermediate and low virulence. Molecular analysis showed large genetic similarity between all isolates and did not separate armadillos from clinical isolates. This study confirms that armadillos harbor pathogenic genotypes of P. brasiliensis, probably the same ones that infect man. Thus, the armadillos are very important animals in the epidemiology this disease

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