Subversion strategies of lysosomal killing by intracellular pathogens.

Many pathogenic organisms need to reach either an intracellular compartment or the cytoplasm of a target cell for their survival, replication or immune system evasion. Intracellular pathogens frequently penetrate into the cell through the endocytic and phagocytic pathways (clathrin-mediated endocytosis, phagocytosis and macropinocytosis) that culminates in fusion with lysosomes. However, several mechanisms are triggered by pathogenic microorganisms - protozoan, bacteria, virus and fungus - to avoid destruction by lysosome fusion, such as rupture of the phagosome and thereby release into the cytoplasm, avoidance of autophagy, delaying in both phagolysosome biogenesis and phagosomal maturation and survival/replication inside the phagolysosome. Here we reviewed the main data dealing with phagosome maturation and evasion from lysosomal killing by different bacteria, protozoa, fungi and virus.

Oxidative stress induced by Pollonein-LAAO, a new L-amino acid oxidase from Bothrops moojeni venom, prompts prostate tumor spheroid cell death and impairs the cellular invasion process in vitro.

Cancer cells produce abnormal levels of reactive oxygen species (ROS) that contribute to promote their malignant phenotype. In this framework, we hypothesized that the change in ROS concentration above threshold could impair key events of prostate cancer cells (PC-3) progression. Our results demonstrated that Pollonein-LAAO, a new L-amino acid oxidase obtained from Bothrops moojeni venom, was cytotoxic to PC-3 cells in two-dimensional and in tumor spheroid assays. Pollonein-LAAO was able to increase the intracellular ROS generation that culminates in cell death from apoptosis by both intrinsic and extrinsic pathways due to the up-regulation of TP53, BAX, BAD, TNFRSF10B and CASP8. Additionally, Pollonein-LAAO reduced mitochondrial membrane potential and caused G0/G1 phase to delay, due to the up-regulation of CDKN1A and the down-regulation of the expression of CDK2 and E2F. Interestingly, Pollonein-LAAO inhibited critical steps of the cellular invasion process (migration, invasion and adhesion), due to the down-regulation of SNAI1, VIM, MMP2, ITGA2, ITGAV and ITGB3. Furthermore, the Pollonein-LAAO effects were associated with the intracellular ROS production, since the presence of catalase restored the invasiveness of PC-3 cells. In this sense, this study contributes to the potential use of Pollonein-LAAO as ROS-based agent to enhance the current understanding of cancer treatment strategies.

Biochemical characterization and assessment of leishmanicidal effects of a new L-amino acid oxidase from Crotalus durissus collilineatus snake venom (CollinLA AO-I).

This study reports the isolation of CollinLAAO-I, a new L-amino acid oxidase from Crotalus durissus collilineatus snake venom, its biochemical characterization and leishmanicidal potential in Leishmania spp. CollinLAAO-I (63.1 kDa) was successfully isolated with high purity using two chromatographic steps and represents 2.5% of total venom proteins. CollinLAAO-I displayed high enzymatic activity (4262.83 U/mg/min), significantly reducing after 28 days. The enzymatic activity of CollinLAAO-I revealed higher affinity for hydrophobic amino acids such as L-leucine, high enzymatic activity in a wide pH range (6.0-10.0), at temperatures from 0 to 25 °C, and showed complete inhibition in the presence of Na+ and K+. Cytotoxicity assays revealed IC50 of 18.49 and 11.66 µg/mL for Leishmania (L.) amazonensis and Leishmania (L.) infantum, respectively, and the cytotoxicity was completely suppressed by catalase. CollinLAAO-I significantly increased the intracellular concentration of reactive oxygen species (ROS) and reduced the mitochondrial potential of both Leishmania species. Furthermore, CollinLAAO-I decreased the parasite capacity to infect macrophages by around 70%, indicating that even subtoxic concentrations of CollinLAAO-I can interfere with Leishmania vital processes. Thus, the results obtained for CollinLAAO-I provide important support for developing therapeutic strategies against leishmaniasis.

Trypanosoma cruzi infection induces proliferation and impairs migration of a human breast cancer cell line.

Breast cancer is considered the type of cancer that most affects women in the world. The triple negative breast cancer is considered aggressive with poor prognosis. In the 1930s Russian researchers observed that T. cruzi has tropism for tumor cells. Since then, this research field has been subject of a numerous of researches. Here, we proposed to investigate the impact of T. cruzi infection on proliferation and migration of triple negative breast cancer cell line (MDA-MB-231). T. cruzi showed high invasion and multiplication rate in MDA-MB-231 cell line. The infection promoted the multiplication of MDA-MB-231 cell, continuous cell lysis throughout of days of in vitro infection and impaired MDA-MB-231 cell migration. Taken together, these results demonstrated the high susceptibility of MDA-MB-231 cell to T. cruzi and suggested that molecules from T. cruzi may impair host cell migration with potential use to avoid metastasis.

Pentachloropseudilin Treatment Impairs Host Cell Invasion by Trypanosoma cruzi.

Pentachloropseudilin (PClP) is a reversible and allosteric inhibitor of type 1 myosin. Here, we addressed the impact of PClP treatment of Trypanosoma cruzi and mammalian host cell on the parasite migration, cell adhesion and invasion. We observed that PClP was not toxic to either T. cruzi or host cell. Moreover, treatment of T. cruzi with PClP inhabited parasite motility, host cell adhesion and invasion. Treatment of host cell with PClP also impaired parasite invasion probably by decreasing lysosome migration to the entry site of the parasite. Therefore, PClP treatment impaired fundamental processes necessary for a successful T. cruzi infection.

Undetected αKlotho in serum is associated with the most aggressive phenotype of breast cancer.

Klotho, a cellular anti-senescence protein, is related to antitumor actions, growth regulation, proliferation and invasiveness in several types of tumor, including breast cancer. The present study aimed to analyze the serum levels of αKlotho in patients with breast cancer according to histopathological and immunohistochemical variables. A total of 74 patients and 60 healthy controls were recruited. Peripheral blood samples were collected and serum levels were assessed by sandwich ELISA. Clinical and diagnostic data were obtained from medical records and databases of the Clinical Hospital of the Federal University of Uberlândia (Uberlândia, Brazil). The results indicated no difference in the levels of αKlotho between patients and controls (P=0.068); however, the number of patients with breast cancer with undetectable αKlotho was high (n=52). Thus, the variables that were associated with the lowest survival rates were analyzed, relating them to undetectable αKlotho. Among cases of metastatic tumors or tumors with poor differentiation, positive lymph node status and triple-negative status, patients with undetectable αKlotho predominated and had unfavorable overall survival. Due to the significant results obtained in triple-negative patients, an in vitro analysis was performed to determine whether estrogen receptors (ERs) have a role in αKlotho production. Treatment of MCF-7 cells with ER agonists, estradiol (E2) and diarylpropionitrile (DPN), resulted in increases in αKlotho expression and supernatant levels of both agonists, demonstrating a direct association between the ER and Klotho production; of note, the ERß-specific agonist DPN tripled αKlotho expression when compared to E2 (P=0.078). These data suggested that undetectable αKlotho in the serum of patients with breast cancer is related to unfavorable histopathological variables and poor prognosis and ERs possibly have an important role in maintaining adequate quantities of αKlotho.

Chemokines and the extracellular matrix: Set of targets for tumor development and treatment.

The extracellular matrix (ECM) consists of various molecules that support tissue cells, including proteins, fibronectin, laminin, collagen IV, and glycosaminoglycans. In addition to interactions between the ECM and cells, the ECM also interacts with chemokines, and growth factors, and these interactions ensure cell survival, development, differentiation, and migration of both immune system cells and tumor cells. This review provides an overview of the mechanisms of interaction between the ECM and chemokines, focusing on the tumor microenvironment and the modulation of these elements as a target for therapies in several types of cancer.

A comparative study on the leishmanicidal activity of the L-amino acid oxidases BjussuLAAO-II and BmooLAAO-II isolated from Brazilian Bothrops snake venoms.

This study aims to examine whether two L-amino acid oxidases isolated from Bothrops snake venom (SV-LAAOs) were cytotoxic to Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis, two causative agents of leishmaniasis, which is an endemic disease in tropical and subtropical countries. The SV-LAAOs BjussuLAAO-II and BmooLAAO-II were isolated from Bothrops jararacussu and Bothrops moojeni venom, respectively, through a three-step chromatography process that used molecular exclusion, hydrophobic interaction, and affinity columns. BmooLAAO-II is a new SV-LAAO isoform that we isolated in this study. The purified BjussuLAAO-II and BmooLAAO-II had high L-amino acid oxidase-specific activity: 3481.17 and 4924.77 U/mg/min, respectively. Both SV-LAAOs were strongly cytotoxic to the two Leishmania species, even at low concentrations. At the same concentration, BjussuLAAO-II and BmooLAAO-II exerted different cytotoxic effects on the parasites. We reported for the first time that the SV-LAAOs suppressed cell proliferation and altered the mitochondrial membrane potential of the two Leishmania species. Surprisingly, BjussuLAAO-II increased the intracellular reactive oxygen species production only in L. (L.) amazonensis, while BmooLAAO-II increased the intracellular reactive oxygen species production only in L. (V.) braziliensis, indicating that these SV-LAAOs had a certain specificity of action.

The Recombinant Protein Based on Trypanosoma cruzi P21 Interacts With CXCR4 Receptor and Abrogates the Invasive Phenotype of Human Breast Cancer Cells.

Trypanosoma cruzi P21 is a protein secreted by the parasite that plays biological roles directly involved in the progression of Chagas disease. The recombinant protein (rP21) demonstrates biological properties, such as binding to CXCR4 receptors in macrophages, chemotactic activity of immune cells, and inhibiting angiogenesis. This study aimed to verify the effects of rP21 interaction with CXCR4 from non-tumoral cells (MCF-10A) and triple-negative breast cancer cells (MDA-MB-231). Our data showed that the MDA-MB-231 cells expressed higher levels of CXCR4 than did the non-tumor cell lines. Besides, cytotoxicity assays using different concentrations of rP21 showed that the recombinant protein was non-toxic and was able to bind to the cell membranes of both cell lineages. In addition, rP21 reduced the migration and invasion of MDA-MB-231 cells by the downregulation of MMP-9 gene expression. In addition, treatment with rP21 blocked the cell cycle, arresting it in the G1 phase, mainly in MDA-MB-231 cells. Finally, rP21 prevents the chemotaxis and proliferation induced by CXCL12. Our data showed that rP21 binds to the CXCR4 receptors in both cells, downregulates CXCR4 gene expression, and decreases the receptors in the cytoplasm of MDA-MB-231 cells, suggesting CXCR4 internalization. This internalization may explain the desensitization of the receptors in these cells. Thus, rP21 prevents migration, invasion, and progression in MDA-MB-231 cells.

Copaifera spp. oleoresins impair Toxoplasma gondii infection in both human trophoblastic cells and human placental explants.

The combination of pyrimethamine and sulfadiazine is the standard care in cases of congenital toxoplasmosis. However, therapy with these drugs is associated with severe and sometimes life-threatening side effects. The investigation of phytotherapeutic alternatives to treat parasitic diseases without acute toxicity is essential for the advancement of current therapeutic practices. The present study investigates the antiparasitic effects of oleoresins from different species of Copaifera genus against T. gondii. Oleoresins from C. reticulata, C. duckei, C. paupera, and C. pubiflora were used to treat human trophoblastic cells (BeWo cells) and human villous explants infected with T. gondii. Our results demonstrated that oleoresins were able to reduce T. gondii intracellular proliferation, adhesion, and invasion. We observed an irreversible concentration-dependent antiparasitic action in infected BeWo cells, as well as parasite cell cycle arrest in the S/M phase. The oleoresins altered the host cell environment by modulation of ROS, IL-6, and MIF production in BeWo cells. Also, Copaifera oleoresins reduced parasite replication and TNF-α release in villous explants. Anti-T. gondii effects triggered by the oleoresins are associated with immunomodulation of the host cells, as well as, direct action on parasites.

Ruthenium (II) complex cis-[RuII(ŋ2-O2CC7H7O2)(dppm)2]PF6-hmxbato induces ROS-mediated apoptosis in lung tumor cells producing selective cytotoxicity.

Ruthenium complexes have been extensively explored as potential molecules for cancer treatment. Considering our previous findings on the remarkable cytotoxic activity exhibited by the ruthenium (II) complex 3-hydroxy-4-methoxybenzoate (hmxbato)-cis-[RuII(ŋ2-O2CC7H7O2)(dppm)2]PF6 against Leishmania promastigotes and also the similar metabolic characteristics between trypanosomatids and tumor cells, the present study aimed to analyze the anticancer potential of hmxbato against lung tumor cells, as well as the partial death mechanisms involved. Hmxbato demonstrated selective cytotoxicity against A549 lung tumor cells. In addition, this complex at a concentration of 3.8 µM was able to expressively increase the generation of reactive oxygen species (ROS) in tumor cells, causing an oxidative stress that may culminate in: (1) reduction in cellular proliferation; (2) changes in cell morphology and organization patterns of the actin cytoskeleton; (3) cell arrest in the G2/M phase of the cell cycle; (4) apoptosis; (5) changes in the mitochondrial membrane potential and (6) initial DNA damage. Furthermore, we demonstrated that the induction of programmed cell death can occur by the intrinsic apoptotic pathway through the activation of caspases. It is also worth highlighting that hmxbato exhibited predominant actions on A549 tumor cells in comparison to BEAS-2B normal bronchial epithelium cells, which makes this complex an interesting candidate for the design of new drugs against lung cancer.

The Recombinant Form of Trypanosoma cruzi P21 Controls Infection by Modulating Host Immune Response.

Trypanosoma cruzi P21 protein (P21) is a putative secreted and immunomodulatory molecule with potent bioactive properties such as induction of phagocytosis and actin cytoskeleton polymerization. Despite the bioactive properties described so far, the action of P21 on parasite replication in muscle cell lineage or T. cruzi parasitism during acute experimental infection is unclear. We observed that recombinant P21 (rP21) decreased the multiplication of T. cruzi in C2C12 myoblasts, phenomenon associated with greater actin polymerization and IFN-γ and IL-4 higher expression. During experimental infection, lower cardiac nests, inflammatory infiltrate and fibrosis were observed in mice infected and treated with rP21. These results were correlated with large expression of IFN-γ counterbalanced by high levels of IL-10, which was consistent with the lower cardiac tissue injury found in these mice. We have also observed that upon stress, such as that induced by the presence of the IFN-γ cytokine, T. cruzi produced more P21. The effect of P21 in controlling the replication of T. cruzi, may indicate an evolutionary mechanism of survival developed by the parasite. Thus, when subjected to different stress conditions, the protozoan produces more P21, which induces T. cruzi latency in the host organism, enabling the protozoan to evade the host's immune system.

Human B cells infected by Trypanosoma cruzi undergo F-actin disruption and cell death via caspase-7 activation and cleavage of phospholipase Cγ1.

B cells contribute to the immune system in many ways such as antigen presentation to CD4+ T cells, secretion of cytokines and lymphoid tissue organogenesis. Furthermore, they are the only cell type capable of producing immunoglobulins. B cells also account for critical aspects of the resistance against intracellular pathogens. Trypanosoma cruzi is an intracellular parasite that sabotages humoral response by depletion of immature B cells. Polyclonal activation and secretion of non-specific antibodies are also other mechanisms used by T cruzi to evade and subvert the mammalian host immune system, leading to increased parasitemia and susceptibility to Chagas' disease. It remained unclear whether B cell depletion occurs due to direct contact with T. cruzi or results from a global increase in inflammation. Unlike previous reports, we demonstrated in this study that T. cruzi infects human B cells, resulting in parasite-induced activation of caspase-7 followed by proteolytic cleavage of phospholipase Cγ1 and cell death. These data contribute to explain the mechanisms ruling B-cell depletion and evasion of the immune response by T. cruzi.

Human B cells infected by Trypanosoma cruzi undergo F-actin disruption and cell death via caspase-7 activation and cleavage of phospholipase Cgama1

B cells contribute to the immune system in many ways such as antigen presentation to CD4+ T cells, secretion of cytokines and lymphoid tissue organogenesis. Furthermore, they are the only cell type capable of producing immunoglobulins. B cells also account for critical aspects of the resistance against intracellular pathogens. Trypanosoma cruzi is an intracellular parasite that sabotages humoral response by depletion of immature B cells. Polyclonal activation and secretion of non-specific antibodies are also other mechanisms used by T cruzi to evade and subvert the mammalian host immune system, leading to increased parasitemia and susceptibility to Chagas’ disease. It remained unclear whether B cell depletion occurs due to direct contact with T. cruzi or results from a global increase in inflammation. Unlike previous reports, we demonstrated in this study that T. cruzi infects human B cells, resulting in parasite-induced activation of caspase-7 followed by proteolytic cleavage of phospholipase Cgama1 and cell death. These data contribute to explain the mechanisms ruling B-cell depletion and evasion of the immune response by T. cruzi.

Human B cells infected by Trypanosoma cruzi undergo F-actin disruption and cell death via caspase-7 activation and cleavage of phospholipase Cgama1

B cells contribute to the immune system in many ways such as antigen presentation to CD4+ T cells, secretion of cytokines and lymphoid tissue organogenesis. Furthermore, they are the only cell type capable of producing immunoglobulins. B cells also account for critical aspects of the resistance against intracellular pathogens. Trypanosoma cruzi is an intracellular parasite that sabotages humoral response by depletion of immature B cells. Polyclonal activation and secretion of non-specific antibodies are also other mechanisms used by T cruzi to evade and subvert the mammalian host immune system, leading to increased parasitemia and susceptibility to Chagas’ disease. It remained unclear whether B cell depletion occurs due to direct contact with T. cruzi or results from a global increase in inflammation. Unlike previous reports, we demonstrated in this study that T. cruzi infects human B cells, resulting in parasite-induced activation of caspase-7 followed by proteolytic cleavage of phospholipase Cgama1 and cell death. These data contribute to explain the mechanisms ruling B-cell depletion and evasion of the immune response by T. cruzi.

Pentachloropseudilin Impairs Angiogenesis by Disrupting the Actin Cytoskeleton, Integrin Trafficking and the Cell Cycle.

Class 1 myosins (Myo1s) were the first unconventional myosins identified and humans have eight known Myo1 isoforms. The Myo1 family is involved in the regulation of gene expression, cytoskeletal rearrangements, delivery of proteins to the cell surface, cell migration and spreading. Thus, the important role of Myo1s in different biological processes is evident. In this study, we have investigated the effects of pentachloropseudilin (PClP), a reversible and allosteric potent inhibitor of Myo1s, on angiogenesis. We demonstrated that treatment of cells with PClP promoted a decrease in the number of vessels. The observed inhibition of angiogenesis is likely to be related to the inhibition of cell proliferation, migration and adhesion, as well as to alteration of the actin cytoskeleton pattern, as shown on a PClP-treated HUVEC cell line. Moreover, we also demonstrated that PClP treatment partially prevented the delivery of integrins to the plasma membrane. Finally, we showed that PClP caused DNA strand breaks, which are probably repaired during the cell cycle arrest in the G1 phase. Taken together, our results suggest that Myo1s participate directly in the angiogenesis process.

The Deleterious Impact of Interleukin 9 to Hepatorenal Physiology.

IL-9 is a pleiotropic cytokine, recently recognized as belonging to Th9 cells that are involved in various pathologies. We aimed to evaluate the role of IL-9 in the course of hepatic and renal fibrosis. Female C57BL/6 mice were treated subcutaneously with IL-9 10 ng/mouse and 20 ng/mouse for 40 days, alternating every 5 days each application, the negative control of which was treated with PBS and positive control with CCL4. IL-9 demonstrated fibrogenic activity, leading to increased collagen I and III deposition in both liver and kidney, as well as triggering lobular hepatitis. In addition, IL-9 induced an inflammatory response with recruitment of lymphocytes, neutrophils, and macrophages to both organs. The inflammation was present in the region of the portal and parenchymal zone in the liver and in the cortical and medullary zone in the kidney. IL-9 deregulated liver and kidney antioxidant activities. Our results showed that IL-9 was able to promote hepatorenal dysfunction. Moreover, IL-9 poses as a promising target for therapeutic interventions.

Increased ROS generation causes apoptosis-like death: Mechanistic insights into the anti-Leishmania activity of a potent ruthenium(II) complex.

Some metallodrugs that exhibit interesting biological activity contain transition metals such as ruthenium, and have been extensively exploited because of their antiparasitic potential. In previous study, we reported the remarkable anti-Leishmania activity of precursor cis-[RuIICl2(dppm)2], where dppm = bis(diphenylphosphino)methane, and new ruthenium(II) complexes, cis-[RuII(η2-O2CC10H13)(dppm)2]PF6 (bbato), cis-[RuII(η2-O2CC7H7S)(dppm)2]PF6 (mtbato) and cis-[RuII(η2-O2CC7H7O2)(dppm)2]PF6 (hmxbato) against some Leishmania species. In view of the promising activity of the hmxbato complex against Leishmania (Leishmania) amazonensis promastigotes, the present work investigated the possible parasite death mechanism involved in the action of this hmxbato and its precursor. We report, for the first time, that hmxbato and precursor promoted an increase in reactive oxygen species production, depolarization of the mitochondrial membrane, DNA fragmentation, formation of a pre-apoptotic peak, alterations in parasite morphology and formation of autophagic vacuoles. Taken together, our results suggest that these ruthenium complexes cause parasite death by apoptosis. Thus, this work provides relevant knowledge on the activity of ruthenium(II) complexes against L. (L.) amazonensis. Such information will be essential for the exploitation of these complexes as future candidates for cutaneous leishmaniasis treatment.

Increased ROS generation causes apoptosis-like death: mechanistic insights into the anti-Leishmania activity of a potent ruthenium(II) complex

Some metallodrugs that exhibit interesting biological activity contain transition metals such as ruthenium, and have been extensively exploited because of their antiparasitic potential. In previous study, we reported the remarkable anti-Leishmania activity of precursor cis-[RuIICl2(dppm)2], where dppm=bis(diphenylphosphino)methane, and new ruthenium(II) complexes, cis-[RuII(n2-O2CC10H13)(dppm)2]PF6 (bbato), cis-[RuII(n2-O2CC7H7S)(dppm)2]PF6 (mtbato) and cis-[RuII(n2-O2CC7H7O2)(dppm)2]PF6 (hmxbato) against some Leishmania species. In view of the promising activity of the hmxbato complex against Leishmania (Leishmania) amazonensis promastigotes, the present work investigated the possible parasite death mechanism involved in the action of this hmxbato and its precursor. We report, for the first time, that hmxbato and precursor promoted an increase in reactive oxygen species production, depolarization of the mitochondrial membrane, DNA fragmentation, formation of a pre-apoptotic peak, alterations in parasite morphology and formation of autophagic vacuoles. Taken together, our results suggest that these ruthenium complexes cause parasite death by apoptosis. Thus, this work provides relevant knowledge on the activity of ruthenium(II) complexes against L. (L.) amazonensis. Such information will be essential for the exploitation of these complexes as future candidates for cutaneous leishmaniasis treatment.

Pentachloropseudilin impairs angiogenesis by disrupting the actin cytoskeleton, integrin trafficking and the cell cycle

Class 1 myosins (Myo1s) were the first unconventional myosins identified and humans have eight known Myo1 isoforms. The Myo1 family is involved in the regulation of gene expression, cytoskeletal rearrangements, delivery of proteins to the cell surface, cell migration and spreading. Thus, the important role of Myo1s in different biological processes is evident. In this study, we have investigated the effects of pentachloropseudilin (PClP), a reversible and allosteric potent inhibitor of Myo1s, on angiogenesis. We demonstrated that treatment of cells with PClP promoted a decrease in the number of vessels. The observed inhibition of angiogenesis is likely to be related to the inhibition of cell proliferation, migration and adhesion, as well as to alteration of the actin cytoskeleton pattern, as shown on a PClP‐treated HUVEC cell line. Moreover, we also demonstrated that PClP treatment partially prevented the delivery of integrins to the plasma membrane. Finally, we showed that PClP caused DNA strand breaks, which are probably repaired during the cell cycle arrest in the G1 phase. Taken together, our results suggest that Myo1s participate directly in the angiogenesis process.