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2.
Mol Cell Biol ; 42(10): e0008122, 2022 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-36073913

RESUMO

The cytosolic tryparedoxin peroxidase (cTXNPx) of Leishmania donovani is a defensive enzyme. Apart from the nonsecretory form, the cTXNPx is released in the spent media of Leishmania cultures and also in the host cell cytosol. The secretory form of the enzyme from the parasite interacts with multiple proteins in the host cell cytosol, the apoptosis-inducing factor (AIF) being one of them. Immunoprecipitation with anti-cTXNPx and anti-AIF antibodies suggests a strong interaction between AIF and cTXNPx. Consequent to parasite invasion, the migration of AIF to the nucleus to precipitate apoptosis is inhibited in the presence of recombinant cTXNPx expressed in the host cell. This inhibition of AIF movement results in lesser host cell death, giving an advantage to the parasite for continued survival. Staurosporine-induced AIF migration to the nucleus was also inhibited in the presence of recombinant cTXNPx in the host cell. Therefore, this study demonstrates the ability of a Leishmania parasite enzyme, cTXNPx, to interfere with the migration of the host AIF protein, providing a survival advantage to the Leishmania parasite.


Assuntos
Fator de Indução de Apoptose , Leishmania donovani , Citosol/metabolismo , Fator de Indução de Apoptose/metabolismo , Estaurosporina/metabolismo , Macrófagos/metabolismo , Apoptose
3.
Front Cell Infect Microbiol ; 11: 694470, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34395309

RESUMO

Pathophysiology of visceral leishmaniasis (VL) is not fully understood and it has been widely accepted that the parasitic components and host immune response both contribute to the perpetuation of the disease. Host alterations during leishmaniasis is a feebly touched area that needs to be explored more to better understand the VL prognosis and diagnosis, which are vital to reduce mortality and post-infection sequelae. To address this, we performed untargeted metabolomics of Leishmania donovani (Ld) infected, uninfected and treated BALB/c mice's tissues and biofluids to elucidate the host metabolome changes using gas chromatography-mass spectrometry. Univariate and multivariate data treatments provided numerous significant differential hits in several tissues like the brain, liver, spleen and bone marrow. Differential modulations were also observed in serum, urine and fecal samples of Ld-infected mice, which could be further targeted for biomarker and diagnostic validations. Several metabolic pathways were found to be upregulated/downregulated in infected (TCA, glycolysis, fatty acids, purine and pyrimidine, etcetera) and treated (arginine, fumaric acid, orotic acid, choline succinate, etcetera) samples. Results also illustrated several metabolites with different pattern of modulations in control, infected and treated samples as well as in different tissues/biofluids; for e.g. glutamic acid identified in the serum samples of infected mice. Identified metabolites include a range of amino acids, saccharides, energy-related molecules, etcetera. Furthermore, potential biomarkers have been identified in various tissues-arginine and fumaric acid in brain, choline in liver, 9-(10) EpOME in spleen and bone marrow, N-acetyl putrescine in bone marrow, etcetera. Among biofluids, glutamic acid in serum, hydrazine and deoxyribose in urine and 3-Methyl-2-oxo pentanoic acid in feces are some of the potential biomarkers identified. These metabolites could be further looked into for their role in disease complexity or as a prognostic marker. The presented profiling approach allowed us to attain a metabolic portrait of the individual tissue/biofluid modulations during VL in the host and represent a valuable system readout for further studies. Our outcomes provide an improved understanding of perturbations of the host metabolome interface during VL, including identification of many possible potential diagnostic and therapeutic targets.


Assuntos
Leishmania donovani , Leishmaniose Visceral , Animais , Metaboloma , Metabolômica , Camundongos , Camundongos Endogâmicos BALB C
4.
Exp Parasitol ; 218: 107987, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32891601

RESUMO

The protozoan parasite Leishmania spp. causes leishmaniases, a group of diseases creating serious health problems in many parts of the world with significant resistance to existing drugs. Insect derived antimicrobial peptides are promising alternatives to conventional drugs against several human disease-causing pathogens because they do not generate resistance. Halictine-2, a novel antimicrobial peptide from the venom of eusocial honeybee, Halictus sexcinctus showed significant anti-leishmanial activity in vitro, towards two life forms of the dimorphic parasite, the free-swimming infective metacyclic promastigotes and the intracellular amastigotes responsible for the systemic infection. The anti-leishmanial activity of the native peptide (P5S) was significantly enhanced by serine to threonine substitution at position 5 (P5T). The peptide showed a propensity to form α-helices after substitution at position-5, conferring amphipathicity. Distinct pores observed on the promastigote membrane after P5T exposure suggested a mechanism of disruption of cellular integrity. Biochemical alterations in the promastigotes after P5T exposure included generation of increased oxygen radicals with mitochondrial Ca2+ release, loss of mitochondrial membrane potential, reduction in total ATP content and increased mitochondrial mass, resulting in quick bioenergetic and chemiosmotic collapse leading to cell death characterized by DNA fragmentation. P5T was able to reduce intracellular amastigote burden in an in vitro model of Leishmania infection but did not alter the proinflammatory cytokines like TNF-α and IL-6. The ability of the P5T peptide to kill the Leishmania parasite with negligible haemolytic activity towards mouse macrophages and human erythrocytes respectively, demonstrates its potential to be considered as a future antileishmanial drug candidate.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Antiprotozoários/farmacologia , Apoptose , Leishmania tropica/efeitos dos fármacos , Substituição de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/química , Antiprotozoários/química , Venenos de Abelha/química , Cálcio/análise , Cálcio/metabolismo , Linhagem Celular , Dicroísmo Circular , Fragmentação do DNA , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Fluorometria , Humanos , Leishmania tropica/ultraestrutura , Leishmaniose Visceral/parasitologia , Macrófagos Peritoneais , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Microscopia Eletrônica de Varredura , Mitocôndrias/química , Tamanho Mitocondrial , Espécies Reativas de Oxigênio/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Superóxidos/isolamento & purificação
5.
Mol Biol Rep ; 47(10): 8049-8060, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32888126

RESUMO

Every disease is an outcome of one or more stress signals which get convened at the interface of the mitochondria. Mitochondria and metabolism are inextricably anchored to each other and a disruption in either can result in the generation of stressors, which can lead to detrimental health consequences. Stowing everything in one frame reflects that the proteins involved in the sensing of stressors are fundamental for the initiation of various pathologies and their detailed study is necessary for proper understanding of disease mechanisms. Sestrins, a class of evolutionarily conserved, stress inducible genes are activated by a wide range of stressors such as oxidative, genotoxic, and metabolic and play a role in cellular homeostasis. In addition, recent reports have highlighted their importance in governing the mitochondrial dynamics and metabolism. However, their spectrum of involvement in various pathologies has not been dissected out very well. This review will focus and discuss the role of Sestrins mainly Sestrin2 and associated nexus in the context of mitochondria, metabolism, and health.


Assuntos
Dano ao DNA , Mitocôndrias/metabolismo , Proteínas Nucleares/metabolismo , Estresse Oxidativo , Humanos
6.
Cell Death Dis ; 10(11): 808, 2019 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-31649242

RESUMO

The importance of autophagy in parasites with a digenetic life cycle like Leishmania spp. is significant. The parasite survives as promastigotes in the insect gut and as immotile amastigotes in mammals. This study demonstrates increased autophagy in Leishmania parasite during progression of in vitro life cycle and upon exposure to stress stimuli like starvation, oxidative stress, and drugs. Autophagy inhibition during stress exposure increased cell death, indicating the importance of autophagy in cellular defense against adverse conditions. Atg8 protein, a homolog of mammalian autophagy protein LC3 is expressed in Leishmania parasite but its function remains unknown. Overexpression of Atg8 (Atg8-OE) rendered the parasites resistant to stress and capable of infecting macrophages in substantial numbers; however, disruption of the Atg8 gene (ΔAtg8) resulting in suppression of Atg8 protein expression, increased susceptibility to stress and reduced the capability to cause infection. A critical event in the Leishmania parasite lifecycle is the differentiation of promastigote forms to the disease causing amastigote forms. The failure of ΔAtg8 parasites lacking Atg8 protein to differentiate into amastigotes, unlike the Atg8-OE and vector-transfected parasites, clearly indicated Atg8 involvement in a crucial event. The inability of ΔAtg8 parasites to infect macrophages in vitro was verified in an in vivo mouse model of leishmaniases where infection could not be induced by the ΔAtg8 parasites. Autophagy is known to be involved in the remodeling of damaged organelles. The accumulation of Atg8 around damaged mitochondria suggested increase of autophagy in the vicinity of the organelle. This buildup was prevented when mitochondria generated reactive oxygen species that were quenched, suggesting them as possible signaling molecules for sensing mitochondrial instability. In summary, our study provides new evidences for a crucial role of Atg8 protein in sustaining Leishmania parasite survival during life cycle and stress exposure, differentiation to amastigotes, and their infective abilities.


Assuntos
Família da Proteína 8 Relacionada à Autofagia/genética , Autofagia/genética , Leishmania donovani/genética , Leishmaniose/genética , Animais , Modelos Animais de Doenças , Humanos , Leishmania donovani/patogenicidade , Leishmaniose/parasitologia , Estágios do Ciclo de Vida/genética , Camundongos , Mitocôndrias/genética , Estresse Oxidativo/genética , Espécies Reativas de Oxigênio/metabolismo
7.
J Immunol ; 202(4): 1163-1175, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30635391

RESUMO

Neutrophils, the essential components of the innate immune system, are recruited in large numbers to the pathogen site of entry. Several pathogens induce neutrophil autophagy; however, function of autophagic events during Leishmania parasite infection remain unknown. In this article, we report a finding that is new, to our knowledge, of how Leishmania-induced human polymorphonuclear neutrophil (hPMN) autophagy regulates the silent mode of parasite transfer to macrophages by influencing the engulfment of infected cells. Leishmania infection induced a time-dependent autophagy increase responsive to block by 3-methyladenine but sensitive to ULK1/2 inhibition only after 3 h. This suggested the prevalence of canonical autophagy during later hours, ULK1/2 inhibition being able to block only canonical autophagy. Interaction of Rubicon and Beclin-1 at 1 h postinfection affirmed the prevalence of noncanonical autophagy during early infection. There was a reduction in macrophage uptake of parasite-exposed hPMNs treated with 3-methyladenine or ULK1/2 inhibitor, suggesting the involvement of both noncanonical and canonical autophagy in neutrophil engulfment. Autophagy inducer rapamycin augmented neutrophil engulfment by macrophages. Redistribution of hPMN surface CD47 encouraged neutrophil uptake. Activation of ERK, phosphoinositide 3-kinase, and NADPH oxidase-mediated reactive oxygen species generation were induced after parasite binding. The lpg1-knockout parasites expressing defective lipophosphoglycan did not induce autophagy, indicating that lipophosphoglycan is necessary for interaction with the neutrophils. Autophagy induction was TLR2/4 independent because the receptor blockade did not interfere with infection-induced autophagy. In summary, the engulfment of neutrophils by the macrophages was influenced by the escalation of hPMN autophagy, which is an important event during Leishmania infection.


Assuntos
Autofagia/imunologia , Leishmania donovani/imunologia , Leishmaniose/imunologia , Neutrófilos/imunologia , Antibacterianos/farmacologia , Autofagia/efeitos dos fármacos , Voluntários Saudáveis , Humanos , Leishmania donovani/efeitos dos fármacos , Leishmaniose/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Neutrófilos/efeitos dos fármacos , Sirolimo/farmacologia
8.
FEBS J ; 285(7): 1326-1345, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29436167

RESUMO

Differential utilization of metabolites and metabolic plasticity can confer adaptation to cancer cells under metabolic stress. Glutamine is one of the vital and versatile nutrients that cancer cells consume avidly for their proliferation, and therefore mechanisms related to glutamine metabolism have been identified as targets. Recently, sestrin2 (SESN2), a stress-inducible protein, has been reported to regulate survival in glutamine-depleted cancer cells; based on this, we explored if SESN2 could regulate glutamine metabolism during glucose starvation. This report highlights the role of SESN2 in the regulation of glutamine-dependent activation of the mitochondrial biogenesis marker peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) under glucose scarcity in liver cancer cells (HepG2). We demonstrate that down-regulation of SESN2 induces a decrease in the levels of intracellular glutamine and PGC-1α under glucose deprivation, concomitant with a decline in cell survival, but no effect was observed on the invasive or migration potential of the cells. Under similar metabolic conditions, SESN2 forms a complex with c-Jun N-terminal kinase (JNK) and forkhead box protein O1 (FOXO1). Absence of SESN2 or inhibition of JNK reduces nuclear translocation of FOXO1, consequently causing transcriptional inhibition of PGC-1α. Notably, our observations demonstrate a reduction in cell viability under high glutamine and low glucose conditions during SESN2 down-regulation that could be rescued on JNK inhibition. To recover from acetaminophen-induced mitochondrial damage, SESN2 was crucial for glutamine-mediated activation of PGC-1α in HepG2 cells. Collectively, we demonstrate a novel role of SESN2 in mediating activation of PGC-1α by modulating glutamine metabolism that facilitates cancer cell survival under glucose-limited metabolic conditions.


Assuntos
Glutamina , Neoplasias Hepáticas , Proteínas Nucleares/metabolismo , Western Blotting , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Glucose/farmacologia , Glutamina/química , Células Hep G2 , Humanos , Neoplasias Hepáticas/fisiopatologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transcrição Gênica/efeitos dos fármacos
9.
Sci Rep ; 8(1): 615, 2018 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-29330382

RESUMO

Mitophagy, the selective degradation of mitochondria by autophagy, is crucial for the maintenance of healthy mitochondrial pool in cells. The critical event in mitophagy is the translocation of cytosolic Parkin, a ubiquitin ligase, to the surface of defective mitochondria. This study elucidates a novel role of SESN2/Sestrin2, a stress inducible protein, in mitochondrial translocation of PARK2/Parkin during mitophagy. The data demonstrates that SESN2 downregulation inhibits BECN1/Beclin1 and Parkin interaction, thereby preventing optimum mitochondrial accumulation of Parkin. SESN2 interacts with ULK1 (unc-51 like kinase 1) and assists ULK1 mediated phosphorylation of Beclin1 at serine-14 position required for binding with Parkin prior to mitochondrial translocation. The trigger for SESN2 activation and regulation of Parkin translocation is the generation of mitochondrial superoxide. Scavenging of mitochondrial superoxide lower the levels of SESN2, resulting in retardation of Parkin translocation. Importantly, we observe that SESN2 mediated cytosolic interaction of Parkin and Beclin1 is PINK1 independent but mitochondrial translocation of Parkin is PINK1 dependent. Together, these findings suggest the role of SESN2 as a positive regulator of Parkin mediated mitophagy.


Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mitocôndrias/química , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular , Citosol/metabolismo , Regulação para Baixo , Células HEK293 , Células HeLa , Humanos , Mitocôndrias/metabolismo , Mitofagia , Fosforilação , Transporte Proteico , Superóxidos/metabolismo
10.
Mol Cell Biochem ; 446(1-2): 1-9, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29294217

RESUMO

Sestrins are evolutionary conserved stress-inducible genes which regulate the axis of cell survival and cell death. Suppression of Sestrin 2 (SESN2) has been linked with increase in oxidative stress and cell death but mechanistic details related to regulation of SESN2 during mitochondrial damage remain unknown. Our study shows that prolonged CCCP-induced mitochondrial damage decreases SESN2 levels and viability of SH-SY5Y cells while overexpression of SESN2 significantly rescues the viability of cells. Further, we demonstrate that Ring box protein 1 (RBX1) is a novel interactive partner and E3 ligase for SESN2 which mediates its K-48-linked ubiquitination upon extensive mitochondrial damage. Downregulation of RBX1 causes stabilization in levels of SESN2. Notably, silencing of RBX1 expression substantially declines cell death and generation of mitochondrial ROS in response to prolonged mitochondrial damage. Taken together, we suggest that SESN2 is critical to protect cells against detrimental effect of mitochondrial damage and RBX1 is a negative regulator of SESN2 which hampers its stabilization.


Assuntos
Proteínas de Transporte/metabolismo , Mitocôndrias/metabolismo , Proteínas de Neoplasias/metabolismo , Neuroblastoma/metabolismo , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas de Transporte/genética , Morte Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Humanos , Mitocôndrias/genética , Mitocôndrias/patologia , Proteínas de Neoplasias/genética , Neuroblastoma/genética , Neuroblastoma/patologia , Proteínas Nucleares/genética , Ubiquitina-Proteína Ligases/genética
12.
Artigo em Inglês | MEDLINE | ID: mdl-29061756

RESUMO

The parasite Leishmania donovani causes visceral leishmaniasis, a potentially fatal disease. The parasites survive within mammalian macrophages and express a unique set of enzymes, the tryparedoxin peroxidases, for their defense against oxidative stress generated by the host. In this study, we demonstrate different roles of two distinct enzymes, the mitochondrial tryparedoxin peroxidase (mTXNPx) and the cytosolic tryparedoxin peroxidase (cTXNPx), in defending the parasites against mitochondrial and exogenous oxidative stress during infection and drug treatment. Our findings indicate a greater increase in cTXNPx expression in response to exogenous oxidative stress and a higher elevation of mTXNPx expression in response to mitochondrial or endogenous stress created by respiratory chain complex inhibitors. Overexpression of cTXNPx in Leishmania showed improved protection against exogenous stress and enhanced protection against mitochondrial stress in parasites overexpressing mTXNPx. Further, parasites overexpressing cTXNPx infected host cells with increased efficiency at early times of infection compared to control parasites or parasites overexpressing mTXNPx. The mTXNPx-overexpressing parasites maintained higher infection at later times. Higher mTXNPx expression occurred in wild-type parasites on exposure to miltefosine, while treatment with antimony elevated cTXNPx expression. Parasites resistant to miltefosine or antimony demonstrated increased expression of mTXNPx, as well as cTXNPx. In summary, this study provides evidence of distinct roles of the two enzymes defined by virtue of their localization during infection and drug treatment.


Assuntos
Leishmania donovani/patogenicidade , Leishmaniose Visceral/tratamento farmacológico , Peroxidases/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Tartarato de Antimônio e Potássio/farmacologia , Citosol/enzimologia , Resistência a Medicamentos/efeitos dos fármacos , Resistência a Medicamentos/genética , Feminino , Interações Hospedeiro-Parasita , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/genética , Leishmaniose Visceral/parasitologia , Macrófagos Peritoneais/parasitologia , Masculino , Camundongos Endogâmicos BALB C , Proteínas Mitocondriais/metabolismo , Estresse Oxidativo , Peroxidases/genética , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Proteínas de Protozoários/genética , Espécies Reativas de Oxigênio/metabolismo , Tripanossomicidas/farmacologia
13.
Sci Rep ; 7(1): 10488, 2017 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-28874760

RESUMO

Micronutrients are essential for survival and growth for all the organisms including pathogens. In this manuscript, we report that zinc (Zn) chelator N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethylenediamine (TPEN) affects growth and viability of intracellular pathogen Leishmania donovani (LD) by a concentration and time dependent manner. Simultaneous addition of zinc salt reverses the effect of TPEN. Further experiments provide evidence of apoptosis-like death of the parasite due to Zn-depletion. TPEN treatment enhances caspase-like activity suggesting increase in apoptosis-like events in LD. Specific inhibitors of cathepsin B and Endoclease G block TPEN-induced leishmanial death. Evidences show involvement of reactive oxygen species (ROS) potentially of extra-mitochondrial origin in TPEN-induced LD death. Pentavalent antimonials remained the prime source of treatment against leishmaniasis for several decades; however, antimony-resistant Leishmania is now common source of the disease. We also reveal that Zn-depletion can promote apoptosis-like death in antimony-resistant parasites. In summary, we present a new finding about the role of zinc in the survival of drug sensitive and antimony-resistant LD.


Assuntos
Apoptose , Leishmania donovani/metabolismo , Zinco/deficiência , Antimônio/toxicidade , Antiprotozoários/toxicidade , Resistência a Medicamentos , Leishmania donovani/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
14.
Front Immunol ; 7: 456, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27826299

RESUMO

Members of the Bcl-2 family are major regulators of apoptosis in mammalian cells, and hence infection-induced perturbations in their expression could result into elimination of the parasites or creation of a niche favoring survival. In this investigation, we uncover a novel role of host Bcl-2 in sustaining Leishmania donovani infection. A rapid twofold increase in Bcl-2 expression occurred in response to parasite challenge. Downregulation of post infection Bcl-2 increase using siRNA or functional inhibition using Bcl-2 small molecule inhibitors interfered with intracellular parasite survival confirming the necessity of elevated Bcl-2 during infection. An increased nitric oxide (NO) response and reduced parasitic burden was observed upon Bcl-2 inhibition, where restitution of the NO response accounted for parasite mortality. Mechanistic insights revealed a major role of elevated Th2 cytokine IL-13 in parasite-induced Bcl-2 expression via the transcription factor STAT-3, where blocking at the level of IL-13 receptor or downstream kinase JAK-2 dampened Bcl-2 induction. Increase in Bcl-2 was orchestrated through Toll like receptor (TLR)-2-MEK-ERK signaling, and changes in TLR-2 levels affected parasite uptake. In a mouse model of visceral leishmaniasis (VL), Bcl-2 inhibitors partially restored the antimicrobial NO response by at least a twofold increase that resulted in significantly reduced parasite burden. Interestingly, monocytes derived from the peripheral blood of six out of nine human VL subjects demonstrated Bcl-2 expression at significantly higher levels, and sera from these patients showed only marginally quantifiable nitrites. Collectively, our study for the first time reveals a pro-parasitic role of host Bcl-2 and the capacity of host-derived IL-13 to modulate NO levels during infection via Bcl-2. Here, we propose Bcl-2 inhibition as a possible therapeutic intervention for VL.

15.
Autophagy ; 12(10): 1817-1831, 2016 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-27459332

RESUMO

Leishmania is an obligate intracellular parasite that replicates inside phagolysosomes or parasitophorous vacuoles (PV) of the monocyte/macrophage lineage. It reprograms macrophages and produces a metabolic state conducive to successful infection and multiplication. MicroRNAs (miRNAs), a class of small 22 to 24 nucleotide noncoding regulatory RNAs alter the gene expression and consequently proteome output by targeting mRNAs, may play a regulatory role in modulating host cell functions. In the present study, we demonstrate the novel regulatory role of host microRNA, MIR30A-3p in modulation of host cell macroautophagy/autophagy after infection with L. donovani. Our in vitro studies showed that MIR30A-3p expression was significantly enhanced after L. donovani infection in a time-dependent manner. Transient transfection with a MIR30A-3p inhibitor followed by L. donovani infection promoted the autophagic response and decreased the intracellular parasite burden in both THP-1 cells and human monocyte-derived macrophages (HsMDM). BECN1/Beclin 1, the mammalian ortholog of yeast Vps30/Atg6, is a key autophagy-promoting protein that plays a key role in the regulation of cell death and survival. We report BECN1-dependent modulation of host cell autophagy in response to L. donovani infection. Pretreatment of L. donovani-infected macrophages with the MIR30A-3p mimic decreased and with antagomir increased the expression of BECN1 protein. We demonstrate that BECN1 is a potential target of MIR30A-3p and this miRNA negatively regulates BECN1 expression. Our present study reveals for the first time a novel role of MIR30A-3p in regulating autophagy-mediated L. donovani elimination by targeting BECN1. The present study has significant impact for the treatment of visceral leishmaniasis.


Assuntos
Autofagia/genética , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Leishmania donovani/fisiologia , Leishmaniose Visceral/genética , Leishmaniose Visceral/parasitologia , MicroRNAs/genética , Animais , Proteínas Relacionadas à Autofagia/metabolismo , Proteína Beclina-1/metabolismo , Linhagem Celular , Análise por Conglomerados , Regulação da Expressão Gênica , Humanos , Leishmaniose Visceral/patologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Macrófagos/patologia , MicroRNAs/metabolismo , Monócitos/patologia , Parasitos/fisiologia , Enzimas de Conjugação de Ubiquitina/metabolismo , Regulação para Cima/genética
16.
J Cancer Policy ; 6: 33-36, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26568911

RESUMO

In 2011, the National Cancer Institute (NCI, USA) introduced the Provocative Questions (PQ) Initiative, a new approach allowing active researchers to define major unsolved or neglected problems in oncology unaddressed by existing funding. Last year, the U.S. NCI teamed up with the Indian Department of Biotechnology (DBT) to pilot the PQ approach in three cities in India. Workshop outcomes includedthe generation of fundable "PQs" (perplexing questions understudied by the international scientific community), as well as the identification of several non-PQ projects and research-related issues of importance to DBT and other Indian funding groups. The workshops clearly indicated the need to expand beyond crafting "PQs" when considering the best areas for research funding in international settings. Nonetheless, the first set of PQ workshops provided a forum to discuss key issues regarding cancer research in India, including the paucity of cancer research funding, and the lack of relevant human resource training and technology sharing platforms. Continued open debate between researchers, funders and policymakers will be essential to effectively strengthen the cancer research portfolio in India.

17.
J Exp Clin Cancer Res ; 34: 142, 2015 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-26590805

RESUMO

BACKGROUND: Colorectal cancer (CRC) ranks third among the estimated cancer cases and cancer related mortalities in the Western world. Early detection and efficient therapy of CRC remains a major health challenge. Therefore, there is a need to identify novel tumor markers for early diagnosis and treatment of CRC. METHODS: A-kinase anchor protein 4 (AKAP4) gene and protein expression was monitored by quantitative polymerase chain reaction (qPCR), reverse transcription (RT)-PCR and Western blotting in normal colon tissue lysate, normal colon epithelial cells and in colon cancer cell lines viz., Caco-2, COLO205, COLO320DM, HCT-15, HCT116, HT-29, SW480, and SW620. The effect of AKAP4 on cellular growth, migration and invasion abilities was studied using gene silencing approach. The role of AKAP4 in various pathways involved in cell cycle, apoptosis, senescence was investigated in in vitro and in human xenograft mouse model. RESULTS: Our studies showed that AKAP4 gene and protein expression was expressed in all colon cancer cells while no expression was detectable in normal colon cells. Ablation of AKAP4 led to reduced cellular growth, migration, invasion and increased apoptosis and senescence of CRC cells in in vitro assays and tumor growth in human xenograft mouse. Human colon xenograft studies showed a significant decrease in the levels of cyclins B1, D and E and cyclin dependent kinases such as CDK1, CDK2, CDK4 and CDK6. Interestingly, an up-regulation in the levels of p16 and p21 was also observed. Besides, an increase in the levels of pro-apoptotic molecules AIF, APAF1, BAD, BID, BAK, BAX, PARP1, NOXA, PUMA and cyt-C and Caspase 3, 7, 8 and 9 was also found in cancer cells as well as in xenograft tissue sections. However, anti-apoptotic molecules BCL2, Bcl-xL, cIAP2, XIAP, Axin2 and Survivin were down regulated in these samples. Our data also revealed elevated expression of epithelial marker E-cadherin and down regulation of EMT markers N-cadherin, P-cadherin, SLUG, α-SMA, SNAIL, TWIST and Vimentin. Further ablation of AKAP4 resulted in the down regulation of invasion molecules matrix metalloproteinase MMP2, MMP3 and MMP9. CONCLUSION: AKAP4 appears to be a novel CRC-associated antigen with a potential for developing as a new clinical therapeutic target.


Assuntos
Proteínas de Ancoragem à Quinase A/biossíntese , Antígenos de Neoplasias/biossíntese , Biomarcadores Tumorais/biossíntese , Neoplasias Colorretais/genética , Proteínas de Ancoragem à Quinase A/genética , Animais , Antígenos de Neoplasias/genética , Apoptose/genética , Biomarcadores Tumorais/genética , Células CACO-2 , Proliferação de Células/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Regulação Neoplásica da Expressão Gênica , Células HT29 , Humanos , Camundongos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto
18.
FASEB J ; 29(10): 4201-13, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26116701

RESUMO

Parasite lipids can serve as signaling molecules, important membrane components, energy suppliers, and pathogenesis factors critical for survival. Functional roles of lipid changes in response to drug-generated stress in parasite survival remains unclear. To investigate this, Leishmania donovani parasites, the causative agents of kala-azar, were exposed to the antileishmanial agent potassium antimony tartrate (PAT) (half-maximal inhibitory concentration ∼ 284 µg/ml). Analysis of cell extracts using gas chromatography-mass spectrometry showed significant increases in very long-chain fatty acids (VLCFAs) prior to an increase in ergosterol in PAT-treated parasites as compared with vehicle-treated controls. Ergosterol biosynthesis inhibition during PAT treatment decreased cell viability. VLCFA inhibition with specific inhibitors completely abrogated ergosterol upsurge followed by a reduction in cell viability. Following PAT-induced VLCFA increase, an upsurge in reactive oxygen species (ROS) occurred and inhibition of this ROS with antioxidants abrogated ergosterol increase. Genetically engineered parasites expressing low constitutive ergosterol levels showed more susceptibility to PAT as compared with wild-type control cells but ergosterol supplementation during PAT treatment increased cell viability. In conclusion, we propose that during antimony treatment, the susceptibility of parasites is determined by the levels of cellular ergosterol that are regulated by oxidative stress generated by VLCFAs.


Assuntos
Antimônio/farmacologia , Ergosterol/biossíntese , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/metabolismo , Acetamidas/farmacologia , Antiprotozoários/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Fragmentação do DNA/efeitos dos fármacos , Ácidos Graxos/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Engenharia Genética , Hemólise/efeitos dos fármacos , Humanos , Leishmania donovani/genética , Leishmaniose Visceral/parasitologia , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/metabolismo , Espécies Reativas de Oxigênio/metabolismo
19.
Am J Cancer Res ; 5(2): 481-97, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25973292

RESUMO

Nitric oxide (NO) has been shown to be effective in cancer chemoprevention and therefore drugs that help generate NO would be preferable for combination chemotherapy or solo use. This study shows a new evidence of NO as a mediator of acute leukemia cell death induced by fisetin, a promising chemotherapeutic agent. Fisetin was able to kill THP-1 cells in vivo resulting in tumor shrinkage in the mouse xenograft model. Death induction in vitro was mediated by an increase in NO resulting in double strand DNA breaks and the activation of both the extrinsic and the intrinsic apoptotic pathways. Double strand DNA breaks could be reduced if NO inhibitor was present during fisetin treatment. Fisetin also inhibited the downstream components of the mTORC1 pathway through downregulation of levels of p70 S6 kinase and inducing hypo-phosphorylation of S6 Ri P kinase, eIF4B and eEF2K. NO inhibition restored phosphorylation of downstream effectors of mTORC1 and rescued cells from death. Fisetin induced Ca(2+) entry through L-type Ca(2+) channels and abrogation of Ca(2+) influx reduced caspase activation and cell death. NO increase and increased Ca(2+) were independent phenomenon. It was inferred that apoptotic death of acute monocytic leukemia cells was induced by fisetin through increased generation of NO and elevated Ca(2+) entry activating the caspase dependent apoptotic pathways. Therefore, manipulation of NO production could be viewed as a potential strategy to increase efficacy of chemotherapy in acute monocytic leukemia.

20.
Infect Immun ; 83(5): 1853-68, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25690103

RESUMO

Leishmania donovani, a protozoan parasite, is the causative agent of visceral leishmaniasis. It lives and multiplies within the harsh environment of macrophages. In order to investigate how intracellular parasite manipulate the host cell environment, we undertook a quantitative proteomic study of human monocyte-derived macrophages (THP-1) following infection with L. donovani. We used the isobaric tags for relative and absolute quantification (iTRAQ) method and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to compare expression profiles of noninfected and L. donovani-infected THP-1 cells. We detected modifications of protein expression in key metabolic pathways, including glycolysis and fatty acid oxidation, suggesting a global reprogramming of cell metabolism by the parasite. An increased abundance of proteins involved in gene transcription, RNA splicing (heterogeneous nuclear ribonucleoproteins [hnRNPs]), histones, and DNA repair and replication was observed at 24 h postinfection. Proteins involved in cell survival and signal transduction were more abundant at 24 h postinfection. Several of the differentially expressed proteins had not been previously implicated in response to the parasite, while the others support the previously identified proteins. Selected proteomics results were validated by real-time PCR and immunoblot analyses. Similar changes were observed in L. donovani-infected human monocyte-derived primary macrophages. The effect of RNA interference (RNAi)-mediated gene knockdown of proteins validated the relevance of the host quantitative proteomic screen. Our findings indicate that the host cell proteome is modulated after L. donovani infection, provide evidence for global reprogramming of cell metabolism, and demonstrate the complex relations between the host and parasite at the molecular level.


Assuntos
Leishmania donovani/imunologia , Macrófagos/química , Macrófagos/parasitologia , Proteoma/análise , Linhagem Celular , Cromatografia Líquida , Perfilação da Expressão Gênica , Humanos , Immunoblotting , Macrófagos/imunologia , Proteômica , Reação em Cadeia da Polimerase em Tempo Real , Espectrometria de Massas em Tandem
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