Up-regulation of TNF-alpha/NFkB/SIRT1 axis drives aggressiveness and cancer stem cells accumulation in chemoresistant oral squamous cell carcinoma.

Tumor resistance remains an obstacle to successfully treating oral squamous cell carcinoma (OSCC). Cisplatin is widely used as a cytotoxic drug to treat solid tumors, including advanced OSCC, but with low efficacy due to chemoresistance. Therefore, identifying the pathways that contribute to chemoresistance may show new possibilities for improving the treatment. This work explored the role of the tumor necrosis factor-alpha (TNF-alpha)/NFkB signaling in driving the cisplatin resistance of OSCC and its potential as a pharmacological target to overcome chemoresistance. Differential accessibility analysis demonstrated the enrichment of opened chromatin regions in members of the TNF-alpha/NFkB signaling pathway, and RNA-Seq confirmed the upregulation of TNF-alpha/NFkB signaling in cisplatin-resistant cell lines. NFkB was accumulated in cisplatin-resistant cell lines and in cancer stem cells (CSC), and the administration of TNF-alpha increased the CSC, suggesting that TNF-alpha/NFkB signaling is involved in the accumulation of CSC. TNF-alpha stimulation also increased the histone deacetylases HDAC1 and SIRT1. Cisplatin-resistant cell lines were sensitive to the pharmacological inhibition of NFkB, and low doses of the NFkB inhibitors, CBL0137, and emetine, efficiently reduced the CSC and the levels of SIRT1, increasing histone acetylation. The NFkB inhibitors decreased stemness potential, clonogenicity, migration, and invasion of cisplatin-resistant cell lines. The administration of the emetine significantly reduced the tumor growth of cisplatin-resistant xenograft models, decreasing NFkB and SIRT1, increasing histone acetylation, and decreasing CSC. TNF-alpha/NFkB/SIRT1 signaling regulates the epigenetic machinery by modulating histone acetylation, CSC, and aggressiveness of cisplatin-resistant OSCC and the NFkB inhibition is a potential strategy to treat chemoresistant OSCC.

Cephaeline is an inductor of histone H3 acetylation and inhibitor of mucoepidermoid carcinoma cancer stem cells.

AIM: To evaluate the potential use of Cephaeline as a therapeutic strategy to manage mucoepidermoid carcinomas (MEC) of the salivary glands. MATERIAL AND METHODS: UM-HMC-1, UM-HMC-2, and UM-HMC-3A MEC cell lines were used to establish the effects of Cephaeline over tumor viability determined by MTT assay. In vitro wound healing scratch assays were performed to address cellular migration while immunofluorescence staining for histone H3 lysine 9 (H3k9ac) was used to identify the acetylation status of tumor cells upon Cephaeline administration. The presence of cancer stem cells was evaluated by the identification of ALDH enzymatic activity by flow cytometry and through functional assays using in vitro tumorsphere formation. RESULTS: A single administration of Cephaeline resulted in reduced viability of MEC cells along with the halt on tumor growth and cellular migration potential. Administration of Cephaeline resulted in chromatin histone acetylation as judged by the increased levels of H3K9ac and disruption of tumorspheres formation. Interestingly, ALDH levels were increased in UM-HMC-1 and UM-HMC-3A cell lines, while UM-HMC-2 showed a reduced enzymatic activity. CONCLUSION: Cephaeline has shown anti-cancer properties in all MEC cell lines tested by regulating tumor cells' viability, migration, proliferation, and disrupting the ability of cancer cells to generate tumorspheres.

Pharmacological inhibition of translocon is sufficient to alleviate endoplasmic reticulum stress and improve Ca2+ handling and contractile recovery of stunned myocardium.

INTRODUCTION: The function of endoplasmic reticulum (ER), a Ca2+ storage compartment and site of protein folding, is altered by disruption of intracellular homeostasis. Misfolded proteins accumulated in the ER lead to ER stress (ERS), unfolded protein response (UPR) activation and ER Ca2+ loss. Myocardial stunning is a temporary contractile dysfunction, which occurs after brief ischemic periods with minimal or no cell death, being oxidative stress and Ca2+ overload potential underlying mechanisms. Myocardial stunning induces ERS response with negatively impact on the post-ischemic mechanical performance through an unknown mechanism. AIMS: In this study, we explored whether ER Ca2+ efflux through the translocon, a major Ca2+ leak channel, contributes to Ca2+ mishandling and the consequent contractile abnormalities of the stunned myocardium. METHODS: Mechanical performance, cytosolic Ca2+, UPR markers and oxidative state were evaluated in perfused rat/mouse hearts subjected to a brief ischemia followed by reperfusion (I/R) in absence or presence of the translocon inhibitor, emetine (1 µM), comparing its effects with those of the chaperones TUDCA (30 µM) and 4-PBA (3 mM). RESULTS: Emetine treatment precluded the I/R-induced increase in UPR signaling markers and improved the contractile recovery together with a remarkable attenuation in myocardial stiffness when compared to I/R hearts with no drug. This alleviation of I/R-induced mechanical abnormalities was more effective than that obtained with the chemical chaperones, TUDCA and 4-PBA. Moreover, emetine treatment produced a striking improvement in diastolic Ca2+ handling with a partial recovery of the I/R-induced oxidative stress. CONCLUSION: Blocking ER Ca2+ store depletion via translocon suppressed ER stress and improved mechanical performance and diastolic Ca2+ handling of stunned myocardium. Modulation of translocon permeability emerges as a therapeutic approach to face dysfunctional consequences of the I/R injury.

The Trans Golgi Region is a Labile Intracellular Ca2+ Store Sensitive to Emetine.

The Golgi apparatus (GA) is a bona fide Ca2+ store; however, there is a lack of GA-specific Ca2+ mobilizing agents. Here, we report that emetine specifically releases Ca2+ from GA in HeLa and HL-1 atrial myocytes. Additionally, it has become evident that the trans-Golgi is a labile Ca2+ store that requires a continuous source of Ca2+ from either the external milieu or from the ER, to enable it to produce a detectable transient increase in cytosolic Ca2+. Our data indicates that the emetine-sensitive Ca2+ mobilizing mechanism is different from the two classical Ca2+ release mechanisms, i.e. IP3 and ryanodine receptors. This newly discovered ability of emetine to release Ca2+ from the GA may explain why chronic consumption of ipecac syrup has muscle side effects.

A Novel Heat Shock Element (HSE) in Entamoeba histolytica that Regulates the Transcriptional Activation of the EhPgp5 Gene in the Presence of Emetine Drug.

Transcriptional regulation of the multidrug resistance EhPgp5 gene in Entamoeba histolytica is induced by emetine stress. EhPgp5 overexpression alters the chloride-dependent currents that cause trophozoite swelling, diminishing induced programmed cell death (PCD) susceptibility. In contrast, antisense inhibition of P-glycoprotein (PGP) expression produces synchronous death of trophozoites and the enhancement of the biochemical and morphological characteristics of PCD induced by G418. Transcriptional gene regulation analysis identified a 59 bp region at position -170 to -111 bp promoter as putative emetine response elements (EREs). However, insights into transcription factors controlling EhPgp5 gene transcription are missing; to fill this knowledge gap, we used deletion studies and transient CAT activity assays. Our findings suggested an activating motif (-151 to -136 bp) that corresponds to a heat shock element (HSE). Gel-shift assays, UV-crosslinking, binding protein purification, and western blotting assays revealed proteins of 94, 66, 62, and 51 kDa binding to the EhPgp5 HSE that could be heat shock-like transcription factors that regulate the transcriptional activation of the EhPgp5 gene in the presence of emetine drug.

Dorsal medial prefrontal cortex contributes to conditioned taste aversion memory consolidation and retrieval.

The medial prefrontal cortex (mPFC) is known for its role in decision making and memory processing, including the participation in the formation of extinction memories. However, little is known regarding its contribution to aversive memory consolidation. Here we demonstrate that neural activity and protein synthesis are required in the dorsal mPFC for memory formation of a conditioned taste aversion (CTA) task and that this region is involved in the retrieval of recent and remote long-term CTA memory. In addition, both NMDA receptor and CaMKII activity in dorsal mPFC are needed for CTA memory consolidation, highlighting the complexity of mPFC functions.

Natural Plant Alkaloid (Emetine) Inhibits HIV-1 Replication by Interfering with Reverse Transcriptase Activity.

Ipecac alkaloids are secondary metabolites produced in the medicinal plant Psychotria ipecacuanha. Emetine is the main alkaloid of ipecac and one of the active compounds in syrup of Ipecac with emetic property. Here we evaluated emetine's potential as an antiviral agent against Human Immunodeficiency Virus. We performed in vitro Reverse Transcriptase (RT) Assay and Natural Endogenous Reverse Transcriptase Activity Assay (NERT) to evaluate HIV RT inhibition. Emetine molecular docking on HIV-1 RT was also analyzed. Phenotypic assays were performed in non-lymphocytic and in Peripheral Blood Mononuclear Cells (PBMC) with HIV-1 wild-type and HIV-harboring RT-resistant mutation to Nucleoside Reverse Transcriptase Inhibitors (M184V). Our results showed that HIV-1 RT was blocked in the presence of emetine in both models: in vitro reactions with isolated HIV-1 RT and intravirion, measured by NERT. Emetine revealed a strong potential of inhibiting HIV-1 replication in both cellular models, reaching 80% of reduction in HIV-1 infection, with low cytotoxic effect. Emetine also blocked HIV-1 infection of RT M184V mutant. These results suggest that emetine is able to penetrate in intact HIV particles, and bind and block reverse transcription reaction, suggesting that it can be used as anti-HIV microbicide. Taken together, our findings provide additional pharmacological information on the potential therapeutic effects of emetine.

Medial prefrontal cortex is a crucial node of a rapid learning system that retrieves recent and remote memories.

The neocortex is thought to be a distributed learning system that gradually integrates semantic information into the initial mnemonic representation rapidly formed by the hippocampus after acquisition. Nevertheless, an emerging view suggests that some cortical regions, in particular the medial prefrontal cortex (mPFC), may also have a role during the initial steps of memory consolidation as well as in the recall of recent memories. Here, we show that mPFC plays a critical role during the first few hours of inhibitory avoidance memory consolidation and is necessary for the normal retrieval of both recent and remote memories, supporting the idea that involvement of neocortical areas in memory processing is not restricted to the late post-training consolidation phase.

On the role of retrosplenial cortex in long-lasting memory storage.

The retrosplenial cortex (RSC) is involved in a range of cognitive functions. However, its precise involvement in memory processing is unknown. Pharmacological and behavioral experiments demonstrate that protein synthesis and c-Fos expression in the anterior part of RSC (aRSC) are necessary late after training to maintain for many days a fear-motivated memory. Long-lasting memory storage is regulated by D1/D5 dopamine receptors in aRSC and depends on the functional interplay between dorsal hippocampus and aRSC. These results suggest that the RSC recapitulates some of the molecular events that occur in the hippocampus to maintain memory trace over time.

Comportamiento clínico-terapéutico de Fasciola hepatica en una serie de 87 pacientes / Clinical and therapeutic behaviour of Fasciola hepatica in a series of 87 patients

Introducción: la fasciolosis, por Fasciola hepatica, muestra a escala mundial un incremento en la incidencia de enfermos en los últimos años. Cuba se encuentra entre aquellos países donde se reportan casos esporádicos y algunos brotes epidémicos. Objetivo: describir el comportamiento clínico-terapéutico de esta trematodiosis de trasmisión digestiva en una serie de 87 pacientes ingresados en el Instituto de Medicina Tropical "Pedro Kourí" desde enero de 1996 a diciembre de 2005. Método: los pacientes se dividieron en 2 grupos atendiendo al fármaco prescrito, dihidroemetina o triclabendazol. Se recogieron las variables clínicas al inicio del diagnóstico y 90 d después del tratamiento; se hallaron las medias y la desviación estándar. Resultados: el sexo masculino predominó discretamente con 54 por ciento en nuestra serie de pacientes ingresados en el servicio de medicina tropical del instituto. La ingestión de berro (Nasturtium officinale) estuvo presente en casi la mitad de los pacientes. El dolor abdominal, fiebre y astenia resultaron los síntomas de mayor frecuencia. El triclabendazol y la dihidroemetina fueron útiles en el tratamiento. Conclusiones: se comprobó la utilidad de los exámenes de laboratorio en el diagnóstico y seguimiento de los enfermos. Los antiparasitarios dihidroemetina y triclabendazol resultaron efectivos a las dosis utilizadas con efectos adversos menores.

Introduction: in the last few years, the Incidence rate of fascioliosis caused by Fasciola hepatica has increased worldwide. Cuba is one of the countries that have reported sporadic cases and also some outbreaks of fasciolosis. Objective: to describe clinical and therapeutic features of this trematodiasis of digestive transmission found in 87 patients, who had been admitted to "Pedro Kourí" Institute of Tropical Medicine from January 1996 to December 2005. Methods: patients were divided into 2 groups according to the prescribed drug, that is, triclabendazole and dihydroemetine. The clinical variables were collected at the time of diagnosis and 90 days after treatment; the means and the standard deviation were estimated. Results: males was slightly predominant (54) in our series of patients admitted to the institute service. Consumption of watercress (Nasturtium officinale) was found in almost half of the patients. Abdominal pain, fever and malaise were the most frequent symptoms. Both drugs were useful to treat F. hepatica. Conclusions: this study showed the usefulness of lab tests for diagnosis and follow-up of patients after treatment. The anti-parasitic drugs dihydroemetine and triclabendazole proved to be effective at the prescribed doses in this research with minor adverse effects.

Human fascioliasis in Argentina: retrospective overview, critical analysis and baseline for future research.

In Argentina, human fascioliasis has never been adequately analysed, although having a physiography, climate, animal prevalences and lymnaeids similar to those of countries where the disease is endemic such as Bolivia, Peru and Chile. We performed a literature search identifying 58 reports accounting for 619 cases, involving 13 provinces, their majority (97.7%) from high altitudes, in central mountainous areas and Andean valleys, concentrated in Cordoba (430 cases), Catamarca (73), San Luis (29) and Mendoza (28), the remaining provinces being rarely affected. This distribution does not fit that of animal fascioliasis. Certain aspects (higher prevalence in females in a local survey, although a trend non-significant throughout Argentina) but not others (patient's age 3-95 years, mean 37.1 years) resemble human endemics in Andean countries, although the lack of intensity studies and surveys in rural areas does not allow for an adequate evaluation. Human infection occurs mainly in January-April, when higher precipitation and temperatures interact with field activities during summer holidays. A second June peak may be related to Easter holidays. The main risk factor appears to be wild watercress ingestion (214) during recreational, weekend outings or holiday activities, explaining numerous family outbreaks involving 63 people and infection far away from their homes. Diagnosis mainly relied on egg finding (288), followed by serology (82), intradermal reaction (63), surgery (43), and erratic fluke observation (6). The number of fascioliasis-hydatidosis co-infected patients (14) is outstanding. Emetine appears as the drug most used (186), replaced by triclabendazole in recent years (21). Surgery reports are numerous (27.0%). A long delay in diagnosis (average almost 3.5 years) and high lithiasis proportion suggest that many patients are frequently overlooked and pose a question mark about fascioliasis detection in the country. High seroprevalences found in recent random surveys suggest human endemic situations. This analysis highlights that human fascioliasis may have been overlooked in the past and its real epidemiological situation in high risk rural, mainly altitudinal areas, may currently be underestimated. Results provide a valuable baseline on which to design appropriate multidisciplinary studies on humans, animals and lymnaeids to assess up to which level and in which areas, human fascioliasis may represent a health problem in Argentina.

[Clinical and therapeutic behaviour of Fasciola hepatica in a series of 87 patients]. / Comportamiento clínico-terapéutico de Fasciola hepatica en una serie de 87 pacientes.

INTRODUCTION: in the last few years, the Incidence rate of fascioliosis caused by Fasciola hepatica has increased worldwide. Cuba is one of the countries that have reported sporadic cases and also some outbreaks of fasciolosis. OBJECTIVE: to describe clinical and therapeutic features of this trematodiasis of digestive transmission found in 87 patients, who had been admitted to "Pedro Kourí" Institute of Tropical Medicine from January 1996 to December 2005. METHODS: patients were divided into 2 groups according to the prescribed drug, that is, triclabendazole and dihydroemetine. The clinical variables were collected at the time of diagnosis and 90 days after treatment; the means and the standard deviation were estimated. RESULTS: males was slightly predominant (54) in our series of patients admitted to the institute service. Consumption of watercress (Nasturtium officinale) was found in almost half of the patients. Abdominal pain, fever and malaise were the most frequent symptoms. Both drugs were useful to treat F. hepatica. CONCLUSIONS: this study showed the usefulness of lab tests for diagnosis and follow-up of patients after treatment. The anti-parasitic drugs dihydroemetine and triclabendazole proved to be effective at the prescribed doses in this research with minor adverse effects.

Molecular phylogeography of Carapichea ipecacuanha, an amphitropical shrub that occurs in the understory of both semideciduous and evergreen forests.

The medicinal shrub Carapichea ipecacuanha (ipecac) is an amphitropic species with three disjunct areas of distribution. In the Brazilian Atlantic and Amazonian ranges, the species was associated mostly with the understory of seasonal semideciduous forests, whereas in the Central American-Colombian range, the species occurred in the understory of moist evergreen forests. We examined the phylogeographic structure of ipecac using chloroplast trnT-trnL and nuclear internal transcribed spacer (ITS) sequences from 120 and 46 specimens, respectively. To complement existing data on root alkaloid profiles, we used high-performance liquid chromatography to assess the levels of emetine and cephaeline in 33 specimens from the two Brazilian ranges. The three ranges shared neither nuclear nor chloroplast haplotypes. The phylogeographic structures showed an uneven distribution of genetic diversity, sharp breaks and high levels of genetic differentiation among ranges. Our results suggest that the extant populations are descendents of at least four distinct ancestral lineages. The Atlantic ipecacs showed higher levels of genetic diversity than ipecacs from the other two ranges; it is likely that they derive from two ancestral lineages, with long-term persistence in that region. The Amazonian ipecacs were monomorphic with respect to the ITS and cpDNA sequences, which supports the view that there was a recent expansion from a single parental source after a strong genetic bottleneck. The existence of a fourth distinct lineage is apparent from the high levels of genetic and chemical differentiation that we identified in the Central American-Columbian ipecacs.

Infusion of protein synthesis inhibitors in the entorhinal cortex blocks consolidation but not reconsolidation of object recognition memory.

Memory consolidation and reconsolidation require the induction of protein synthesis in some areas of the brain. Here, we show that infusion of the protein synthesis inhibitors anisomycin, emetine and cycloheximide in the entorhinal cortex immediately but not 180 min or 360 min after training in an object recognition learning task hinders long-term memory retention without affecting short-term memory or behavioral performance. Inhibition of protein synthesis in the entorhinal cortex after memory reactivation involving either a combination of familiar and novel objects or two familiar objects does not affect retention. Our data suggest that protein synthesis in the entorhinal cortex is necessary early after training for consolidation of object recognition memory. However, inhibition of protein synthesis in this cortical region after memory retrieval does not seem to affect the stability of the recognition trace.

[Hepatic fasciolasis diagnosed in state phase]. / Fasciolosis hepática diagnosticada en fase de estado.

INTRODUCTION: Hepatic fasciolosis is a zoonosis that accidentally can invade the human. REPORT OF A CASE: 62 years old male, farmer, lives in a rural community in Tehuacan, Puebla, Mexico. His living space is not provided with running water nor drainage. He has contact with sheep and bovines. Started presenting symptoms two years before. Suffered from myalgia, joint pain, fever of 38 degrees C and epigastric pain that radiated the hypocondrium and the right shoulder. He had diarrhea five times in 24 hours as well as lack of appetite that lead to a weight loss of 20 kilograms in two years. He was hospitalized and the physical examination revealed diminished muscular mass, right hypocondrium pain and hepatomegaly of 3 cm below costal margin. He said he ate watercress (Nasturium officinalis) two or three times a week. Blood test revealed erythrocytes of 3.6 x 105 mm3; hemoglobin of 11.9 g/dL; hematocrit of 30%; leukocytes 8950 mm3; neutrophils 65%; lymphocytes of 30%; eosinophils of 3%; monocytes of 1% and basophiles of 1%. Globular sedimentation was 83 mm and hemoglobin concentration was 33. Liver test results were normal and mycobacterium in fecal samples was negative, but stool detection tests revealed eggs of Fasciola hepatica. The diagnosis was hepatic fasciolosis in its biliar stage. Dehidrohemetine (1.5 mg/kg) was administered during 10 days. Symptoms disappeared within 48 hours. CONCLUSION: The lack of knowledge about fasciolosis makes it hard to diagnose it. The publication of case reports must help to facilitate its diagnosis.

Entamoeba histolytica EhPgp5 transcriptional activation depends on putative emetine response elements.

The multidrug resistance EhPgp5 gene promoter is active in drug resistant clone C2 trophozoites and its activity increases when trophozoites are cultured in the presence of emetine, suggesting that the EhPgp5 gene shows an inducible drug dependent mechanism. We analyzed different promoter fragments to detect those regions that activate transcription in the presence of emetine. Trophozoites were transfected with p375Pgp5, p259Pgp5, p187Pgp5, and p76Pgp5 plasmids and incubated with different emetine concentrations. p375Pgp5 and p259Pgp5 plasmids were able to drive CAT expression in A and C2 trophozoites only in the presence of emetine. CAT activity was turned off in the absence of drug. Interestingly, no CAT activity was detected in the presence or in the absence of emetine with p187Pgp5 plasmid in which 59 bp were deleted at the 5' end of the EhPgp5 minimal promoter (p259Pgp5). These results suggest that the overexpression of the EhPgp5 gene is a consequence of transcriptional activation of the gene promoter by putative drug responsive elements, located within the -111 to -170 bp of the transcription initiation site.

Histopathology of cutaneous amebiasis.

Cutaneous amebiasis (CA) is the manifestation in the skin and underlying soft tissues of the pathogenic properties of Entamoeba histolytica, which may be the only expression of the infection or may be associated with disease in other organs. So far, there have been only isolated case reports on this disease. We herein report the histopathologic findings on a series of seven cases, six adults and one child, of CA. The most common findings include ulcers, areas of necrosis, mixed inflammatory infiltrates, and the presence of trophozoites, the invasive form of the parasite. CA is a very rare and severe disease, it is progressive and destructive; erythrophagocytosis, a microscopic sign of pathogenicity, is always seen in CA.

[Fascioliasis: diagnosis, epidemiology and treatment]. / Fascioliasis. Diagnóstico, epidemiología y tratamientos.

Fascioliasis is a trematode, disease of liver and bile ducts of sheep, cattle, and other ruminants throughout the world that is caused by the fluke, Fasciola hepatica. Human infection has been reported in Mexico, Cuba, Puerto Rico, Chile, Peru, Uruguay, Brazil, Argentina, the US, Europe, eastern Africa, Japan and Australia. The parasite's miracidium invades one of the various Lymnaea water snail hosts. Infection results from ingestion of encysted metacercariae attached to raw watercress (Nasturtium officinale). Symptoms recorded from human cases included irregular fever, epigastric pain and abdominal tenderness, obstructive jaundice and leucocytosis with eosinophilea up to 60%. Specific diagnosis is based on recovery of the eggs in the patient's stool or from biliary tract drainage. Treatment is with emetine hydrochloride given intramusculary. Bithionol is given orally at a dosage of 30-50 mg/kg but on alternate days from 10 to 15 doses. Praziquantel is probably effective. Preventive measures include education of the public on mode of transmission of life cycle of the parasite, and dipping fresh watercress into boiling water for a few sec, or drying suspected watercress.