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Introduction: Heparan sulfate (HS) in the vascular endothelial glycocalyx (eGC) is a critical regulator of blood vessel homeostasis. Trauma results in HS shedding from the eGC, but the impact of trauma on HS structural modifications that could influence mechanisms of vascular injury and repair has not been evaluated. Moreover, the effect of eGC HS shedding on endothelial cell (EC) homeostasis has not been fully elucidated. The objectives of this work were to characterize the impact of trauma on HS sulfation and determine the effect of eGC HS shedding on the transcriptional landscape of vascular ECs. Methods: Plasma was collected from 25 controls and 49 adults admitted to a level 1 trauma center at arrival and 24 h after hospitalization. Total levels of HS and angiopoietin-2, a marker of pathologic EC activation, were measured at each time point. Enzymatic activity of heparanase, the enzyme responsible for HS shedding, was determined in plasma from hospital arrival. Liquid chromatography-tandem mass spectrometry was used to characterize HS di-/tetrasaccharides in plasma. In vitro work was performed using flow conditioned primary human lung microvascular ECs treated with vehicle or heparinase III to simulate human heparanase activity. Bulk RNA sequencing was performed to determine differentially expressed gene-enriched pathways following heparinase III treatment. Results: We found that heparanase activity was increased in trauma plasma relative to controls, and HS levels at arrival were elevated in a manner proportional to injury severity. Di-/tetrasaccharide analysis revealed lower levels of 3-O-sulfated tetramers with a concomitant increase in ΔIIIS and ΔIIS disaccharides following trauma. Admission levels of total HS and specific HS sulfation motifs correlated with 24-h angiopoietin-2 levels, suggesting an association between HS shedding and persistent, pathological EC activation. In vitro pathway analysis demonstrated downregulation of genes that support cell junction integrity, EC polarity, and EC senescence while upregulating genes that promote cell differentiation and proliferation following HS shedding. Discussion: Taken together, our findings suggest that HS cleavage associated with eGC injury may disrupt homeostatic EC signaling and influence biosynthetic mechanisms governing eGC repair. These results require validation in larger, multicenter trauma populations coupled with in vivo EC-targeted transcriptomic and proteomic analyses.
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Heparanase is upregulated during the progression of most cancers and via its enzyme activity promotes extracellular matrix degradation, angiogenesis and cell migration. Heparanase expression is often associated with enhanced tumor aggressiveness and chemoresistance. We previously demonstrated that increased heparanase expression in tumor cells enhances secretion and alters the composition of tumor-released exosomes. In the present study, we discovered that extracellular vesicles (EVs) secreted by human multiple myeloma cells growing in hypoxic conditions exhibited elevated levels of heparanase cargo compared to EVs from cells growing in normoxic conditions. When macrophages (RAW 264.7 monocyte/macrophage-like cells) were exposed to EVs released by tumor cells growing in either hypoxic or normoxic conditions, macrophage migration and invasion was elevated by EVs from hypoxic conditions. The elevated invasion of macrophages was blocked by a monoclonal antibody that inhibits heparanase enzyme activity. Moreover, the heparanase-bearing EVs from hypoxic cells greatly enhanced endothelial cell tube formation consistent with the known role of heparanase in promoting angiogenesis. EVs from hypoxic tumor cells when compared with EVs from normoxic cells also enhanced cancer stemness properties of both CAG and RPMI 8226 human myeloma cells. Together these data indicate that under hypoxic conditions, tumor cells secrete EVs having an elevated level of heparanase as cargo. These EVs can act on both tumor and non-tumor cells, enhancing tumor progression and tumor cell stemness that likely supports chemoresistance and relapse of tumor.
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Triple negative breast cancer (TNBC), an aggressive and highly metastatic subtype of breast cancer. Glioma-associated oncogene 1 (GLI1) is a transcription factor and effector of the Hedgehog (Hh) signaling pathway, and is predictive of poor survival for TNBC patients. A nanostring DNA Damage Response (DDR) mRNA panel was used to identify GLI1-induced regulation of DDR genes. Western blots, immunohistochemistry and immunofluorescence were used to evaluate protein expression. Colony assays and mammosphere formation assays were utilized to assess survival of cancer cells. Flow cytometry analyses were employed to evaluate changes in the cell cycle profile, and DNA fiber assays were used to analyze alterations in replication dynamics in TNBC cells. The UALCAN portal and Ensemble programs were used for computational analysis of TCGA data. CompuSyn software was used to calculate combination index (CI) values to assess synergism in drug combination experiments. Inhibition of GLI1 in TNBC cells transcriptionally downregulate expression of FANCD2 and its foci formation, and causes a homologous recombination repair (HR) deficiency. As HR-deficient cancer cells are sensitive to PARP-targeted therapies, we evaluated a combination of the GLI1 inhibitor, GANT61, and a PARP inhibitor (olaparib) in TNBC cells. Combination of GANT61 and olaparib elevated DNA damage levels and these drug combinations caused synergistic lethality to TNBC cells. Aberrantly activated GLI1 regulates HR-mediated DNA repair by transcriptionally regulating FANCD2 to overcome chemotherapy-induced replication stress and DNA damage, and it contributes to resistance of TNBC cells to therapeutics.
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Replicación del ADN , Sinergismo Farmacológico , Recombinación Homóloga , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Piridinas/farmacología , Pirimidinas/farmacología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Proteína con Dedos de Zinc GLI1/antagonistas & inhibidores , Apoptosis , Ciclo Celular , Movimiento Celular , Proliferación Celular , Quimioterapia Combinada , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Humanos , Estrés Oxidativo , Pronóstico , Tasa de Supervivencia , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Células Tumorales CultivadasRESUMEN
Raga todi, an Indian classical music raga is emerging as a therapeutic approach in mental health and well being. This study aims to assess the relationship of raga todi and state anxiety at the time of novel coronavirus spread across the globe. 30 young female adults of age 20-28 years were randomly assigned to experimental group and control group of 15 participants each to which a pre and post test of State Anxiety test of STAI-A was administered. Results stated that state anxiety is negatively correlated to the listening of raga todi. Thus, listening to raga todi could be useful in reducing the state anxiety level provoked by a stressful life event like the lethal coronavirus pandemic.
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Ovarian cancer (OC) is one of the most lethal type of cancer in women due to a lack of effective targeted therapies and high rates of treatment resistance and disease recurrence. Recently Poly (ADP-ribose) polymerase inhibitors (PARPi) have shown promise as chemotherapeutic agents; however, their efficacy is limited to a small fraction of patients with BRCA mutations. Here we show a novel function for the Hedgehog (Hh) transcription factor Glioma associated protein 1 (GLI1) in regulation of key Fanconi anemia (FA) gene, FANCD2 in OC cells. GLI1 inhibition in HR-proficient OC cells induces HR deficiency (BRCAness), replication stress and synergistic lethality when combined with PARP inhibition. Treatment of OC cells with combination of GLI1 and PARP inhibitors shows enhanced DNA damage, synergy in cytotoxicity, and strong in vivo anticancer responses.
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Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteínas Hedgehog/metabolismo , Recombinación Homóloga/fisiología , Neoplasias Ováricas/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Proteína con Dedos de Zinc GLI1/metabolismo , Animales , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Femenino , Proteínas Hedgehog/antagonistas & inhibidores , Proteínas Hedgehog/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones Transgénicos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Ftalazinas/farmacología , Ftalazinas/uso terapéutico , Piperazinas/farmacología , Piperazinas/uso terapéutico , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Piridinas/farmacología , Piridinas/uso terapéutico , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Transcripción Genética/efectos de los fármacos , Transcripción Genética/fisiología , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Proteína con Dedos de Zinc GLI1/antagonistas & inhibidores , Proteína con Dedos de Zinc GLI1/genéticaRESUMEN
Heparanase (HPSE) is an endoglycosidase that cleaves heparan sulfate and has been shown in various cancers to promote metastasis, angiogenesis, osteolysis, and chemoresistance. Although heparanase is thought to act predominantly extracellularly or within the cytoplasm, it is also present in the nucleus, where it may function in regulating gene transcription. Using myeloma cell lines, we report here that heparanase enhances chromatin accessibility and confirm a previous report that it also upregulates the acetylation of histones. Employing the Multiple Myeloma Research Foundation CoMMpass database, we demonstrate that patients expressing high levels of heparanase display elevated expression of proteins involved in chromatin remodeling and several oncogenic factors compared to patients expressing low levels of heparanase. These signatures were consistent with the known function of heparanase in driving tumor progression. Chromatin opening and downstream target genes were abrogated by inhibition of heparanase. Enhanced levels of heparanase in myeloma cells led to a dramatic increase in phosphorylation of PTEN, an event known to stabilize PTEN, leading to its inactivity and loss of tumor suppressor function. Collectively, this study demonstrates that heparanase promotes chromatin opening and transcriptional activity, some of which likely is through its impact on diminishing PTEN tumor suppressor activity.
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Cromatina/genética , Regulación Neoplásica de la Expresión Génica , Glucuronidasa/genética , Mieloma Múltiple/genética , Fosfohidrolasa PTEN/genética , Acetilación , Línea Celular Tumoral , Núcleo Celular/enzimología , Núcleo Celular/genética , Cromatina/metabolismo , Genes Supresores de Tumor , Glucuronidasa/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Mieloma Múltiple/enzimología , Mieloma Múltiple/patología , Fosfohidrolasa PTEN/metabolismo , Regulación hacia ArribaRESUMEN
Chemotherapy involves the use of multiple cytotoxic or cytostatic drugs acting by various mechanisms to kill or arrest the growth of cancer cells. Chemotherapy remains the most utilized approach for controlling cancer. Emerging evidence indicates that cancer cells activate various pro-survival mechanisms to cope with chemotherapeutic stress. These mechanisms persist during treatment and often help orchestrate tumor regrowth and patient relapse. Exosomes due to their nature of carrying and transferring multiple biologically active components have emerged as key players in cancer pathogenesis. Recent data demonstrates that chemotherapeutic stress enhances the secretion and alters the cargo carried by exosomes. These altered exosomes, which we refer to as chemoexosomes, are capable of transferring cargo to target tumor cells that can enhance their chemoresistance, increase their metastatic behavior and in certain cases even aid in endowing tumor cells with cancer stem cell-like properties. This mini-review summarizes the recent developments in our understanding of the impact chemoexosomes have on tumor survival and progression.
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Resistencia a Antineoplásicos , Exosomas/metabolismo , Neoplasias/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias/tratamiento farmacológico , Células Madre Neoplásicas/metabolismoRESUMEN
Both heparanase and syndecan-1 are known to be present and active in disease pathobiology. An important feature of syndecan-1 related to its role in pathologies is that it can be shed from the surface of cells as an intact ectodomain composed of the extracellular core protein and attached heparan sulfate and chondroitin sulfate chains. Shed syndecan-1 remains functional and impacts cell behavior both locally and distally from its cell of origin. Shedding of syndecan-1 is initiated by a variety of stimuli and accomplished predominantly by the action of matrix metalloproteinases. The accessibility of these proteases to the core protein of syndecan-1 is enhanced, and shedding facilitated, when the heparan sulfate chains of syndecan-1 have been shortened by the enzymatic activity of heparanase. Interestingly, heparanase also enhances shedding by upregulating the expression of matrix metalloproteinases. Recent studies have revealed that heparanase-induced syndecan-1 shedding contributes to the pathogenesis and progression of cancer and viral infection, as well as other septic and non-septic inflammatory states. This review discusses the heparanase/shed syndecan-1 axis in disease pathogenesis and progression, the potential of targeting this axis therapeutically, and the possibility that this axis is widespread and of influence in many diseases.
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Progresión de la Enfermedad , Glucuronidasa/metabolismo , Neoplasias/metabolismo , Sindecano-1/metabolismo , Virosis/metabolismo , Humanos , Neoplasias/patología , Virosis/patologíaRESUMEN
Phosphofurin acidic cluster sorting protein-1 (PACS-1) is a multifunctional membrane traffic regulator that plays important roles in organ homeostasis and disease. In this study, we elucidate a novel nuclear function for PACS-1 in maintaining chromosomal integrity. PACS-1 progressively accumulates in the nucleus during cell cycle progression, where it interacts with class I histone deacetylases 2 and 3 (HDAC2 and HDAC3) to regulate chromatin dynamics by maintaining the acetylation status of histones. PACS-1 knockdown results in the proteasome-mediated degradation of HDAC2 and HDAC3, compromised chromatin maturation, as indicated by elevated levels of histones H3K9 and H4K16 acetylation, and, consequently, increased replication stress-induced DNA damage and genomic instability.
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Cromatina/fisiología , Inestabilidad Genómica , Histona Desacetilasa 1/metabolismo , Histona Desacetilasas/metabolismo , Proteínas de Transporte Vesicular/fisiología , Ciclo Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Citosol/metabolismo , Replicación del ADN , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica , Proteínas de Transporte Vesicular/genéticaRESUMEN
Heparanase is known to enhance the progression of many cancer types and is associated with poor patient prognosis. We recently reported that after patients with multiple myeloma were treated with high dose chemotherapy, the tumor cells that emerged upon relapse expressed a much higher level of heparanase than was present prior to therapy. Because tumor cells having stemness properties are thought to seed tumor relapse, we investigated whether heparanase had a role in promoting myeloma stemness. When plated at low density and grown in serum-free conditions that support survival and expansion of stem-like cells, myeloma cells expressing a low level of heparanase formed tumor spheroids poorly. In contrast, cells expressing a high level of heparanase formed significantly more and larger spheroids than did the heparanase low cells. Importantly, heparanase-low expressing cells exhibited plasticity and were induced to exhibit stemness properties when exposed to recombinant heparanase or to exosomes that contained a high level of heparanase cargo. The spheroid-forming heparanase-high cells had elevated expression of GLI1, SOX2 and ALDH1A1, three genes known to be associated with myeloma stemness. Inhibitors that block the heparan sulfate degrading activity of heparanase significantly diminished spheroid formation and expression of stemness genes implying a direct role of the enzyme in regulating stemness. Blocking the NF-κB pathway inhibited spheroid formation and expression of stemness genes demonstrating a role for NF-κB in heparanase-mediated stemness. Myeloma cells made deficient in heparanase exhibited decreased stemness properties in vitro and when injected into mice they formed tumors poorly compared to the robust tumorigenic capacity of cells expressing higher levels of heparanase. These studies reveal for the first time a role for heparanase in promoting cancer stemness and provide new insight into its function in driving tumor progression and its association with poor prognosis in cancer patients.
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Regulación hacia Abajo , Glucuronidasa/genética , Mieloma Múltiple/patología , Células Madre Neoplásicas/patología , Familia de Aldehído Deshidrogenasa 1/genética , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Exosomas/enzimología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Mieloma Múltiple/genética , Trasplante de Neoplasias , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/enzimología , Retinal-Deshidrogenasa/genética , Factores de Transcripción SOXB1/genética , Esferoides Celulares/citología , Proteína con Dedos de Zinc GLI1/genéticaRESUMEN
The normal female reproductive hormone estrogen has been linked with increased risk of breast and many other forms of cancer. This is largely due to metabolic conversion of estrogens into highly reactive catechol estrogen quinones which can interact with DNA and cause a variety of DNA adducts and lesions. Detection and analysis of these adducts and their associated cellular responses involve complex chemical, enzymatic, and LC-MS based methods, which are both laborious and require specialized expertise and instrumentation. Herein, we show that using a biotin-labeled estradiol allows immunodetection of estrogen-induced DNA adducts by slot blot and single-cell molecular combing and proximity ligation assays. The biotinylated and unlabeled estradiols induced similar levels of DNA single and double strand breaks as measured by comet assays. Using biotinylated estrogen, we further show that estrogens are able to activate the Fanconi anemia-BRCA tumor suppressor pathway and cause DNA strand breaks and oxidatively modified DNA bases as well as gross chromosomal aberrations. Utilization of biotin-labeled estrogens could be a powerful tool to detect estrogen adducts and associated DNA damage, and to track estrogen adduct-induced cellular responses and carcinogenic mechanisms in cultured cells. The techniques presented here allow simple and rapid detection and quantitation of estrogen adducts by slot blot as well as direct visualization on the DNA strand and could pave the way for developing new treatments to protect the genome from the effects of reactive estrogen metabolites. © 2016 Wiley Periodicals, Inc.
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Carcinógenos/metabolismo , Aductos de ADN/metabolismo , Estradiol/química , Estrógenos/toxicidad , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Biotinilación , Células Cultivadas , Aberraciones Cromosómicas , Estrógenos/química , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Células MCF-7 , Análisis de la Célula IndividualRESUMEN
Series of 4-amino-6-(arylamino)-1,3,5-triazine-2-carbohydrazides (3a-e) and N'-phenyl-4,6-bis(arylamino)-1,3,5-triazine-2-carbohydrazides (6a-e), for ease of readership, we will abbreviate our compound names as 'new triazines', have been synthesized, based on the previously reported Rad6B-inhibitory diamino-triazinylmethyl benzoate anticancer agents TZ9 and 4-amino-N'-phenyl-6-(arylamino)-1,3,5-triazine-2-carbohydrazides. Synthesis of the target compounds was readily accomplished in two steps from either bis-aryl/aryl biguanides via reaction of phenylhydrazine or hydrazinehydrate with key 4-amino-6-bis(arylamino)/(arylamino)-1,3,5-triazine-2-carboxylate intermediates. These new triazine derivatives were evaluated for their abilities to inhibit Rad6B ubiquitin conjugation and in vitro anticancer activity against several human cancer cell lines: ovarian (OV90 and A2780), lung (H1299 and A549), breast (MCF-7 and MDA-MB231) and colon (HT29) cancer cells by MTS assays. All the 10 new triazines exhibited superior Rad6B inhibitory activities in comparison to selective Rad6 inhibitor TZ9 that was reported previously. Similarly, new triazines also showed better IC50 values in survival assays of various tumor cell lines. Particularly, new triazines 6a-c, exhibited lower IC50 (3.3-22 µM) values compared to TZ9.
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Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Hidrazinas/farmacología , Triazinas/farmacología , Enzimas Ubiquitina-Conjugadoras/antagonistas & inhibidores , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Hidrazinas/síntesis química , Hidrazinas/química , Estructura Molecular , Relación Estructura-Actividad , Triazinas/síntesis química , Triazinas/química , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Camptothecin (CPT) and its analogues are chemotherapeutic agents that covalently and reversibly link DNA Topoisomerase I to its nicked DNA intermediate eliciting the formation of DNA double strand breaks (DSB) during replication. The repair of these DSB involves multiple DNA damage response and repair proteins. Here we demonstrate that CPT-induced DNA damage promotes functional interactions between BRCA2, FANCD2, Rad18, and Rad51 to repair the replication-associated DSB through homologous recombination (HR). Loss of any of these proteins leads to equal disruption of HR repair, causes chromosomal aberrations and sensitizes cells to CPT. Rad18 appears to function upstream in this repair pathway as its downregulation prevents activation of FANCD2, diminishes BRCA2 and Rad51 protein levels, formation of nuclear foci of all three proteins and recovery of stalled or collapsed replication forks in response to CPT. Taken together this work further elucidates the complex interplay of DNA repair proteins in the repair of replication-associated DSB.
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Camptotecina/farmacología , Roturas del ADN de Doble Cadena , Reparación del ADN , ADN-Topoisomerasas de Tipo I/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Recombinasa Rad51/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Células HCT116 , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , ARN Interferente Pequeño/metabolismo , Transfección , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Ovarian cancer is the fifth most deadly cancer in women in the United States and despite advances in surgical and chemotherapeutic treatments survival rates have not significantly improved in decades. The poor prognosis for ovarian cancer patients is largely due to the extremely high (80%) recurrence rate of ovarian cancer and because the recurrent tumors are often resistant to the widely utilized platinum-based chemotherapeutic drugs. In this study, expression of Rad6, an E2 ubiquitin-conjugating enzyme, was found to strongly correlate with ovarian cancer progression. Furthermore, in ovarian cancer cells Rad6 was found to stabilize ß-catenin promoting stem cell-related characteristics, including expression of stem cell markers and anchorage-independent growth. Cancer stem cells can promote chemoresistance, tumor recurrence and metastasis, all of which are limiting factors in treating ovarian cancer. Thus it is significant that Rad6 overexpression led to increased resistance to the chemotherapeutic drug carboplatin and correlated with tumor cell invasion. These findings show the importance of Rad6 in ovarian cancer and emphasize the need for further studies of Rad6 as a potential target for the treatment of ovarian cancer.
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Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Compuestos de Platino/administración & dosificación , Enzimas Ubiquitina-Conjugadoras/metabolismo , Antineoplásicos/administración & dosificación , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Femenino , Humanos , Células Madre Neoplásicas/efectos de los fármacos , Neoplasias Ováricas/patología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Ascorbate peroxidase (E 1.11.1.11) acts as primary key component of plant defense against photo protection and photo-oxidative stress. Chloroplastic (APX) located in the thylakoid membrane (tAPX) and stroma (sAPX) have been thought to be key regulators of intracellular levels of H2O2. Therefore, it is of interest to study thylakoid membrane bound SlAPX from Solanum lycopersicum (tomato, a fleshy fruit). However, a structure model is not yet solved for tomato thylakoid membrane bound SlAPX. Hence, a homology molecular model of SlAPX6 from S. lycopersicum was constructed using a template structure (PDB ID: 1APX) from Pisum sativum. The model was further assessed using accessible surface area (ASA) calculations to identify surface residues for further characterization of active site regions. We further characterized the active site regions in the enzyme for functional inference. This information provides insights for the understanding of photo protection and photo-oxidative stress tolerant in S. lycopersicum during flower development and fruit ripening.
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The canonical hedgehog (HH) pathway is a multicomponent signaling cascade (HH, protein patched homolog 1 (PTCH1), smoothened (SMO)) that plays a pivotal role during embryonic development through activation of downstream effector molecules, namely glioma-associated oncogene homolog 1 (GLI1), GLI2 and GLI3. Activation of GLIs must be tightly regulated as they modulate target genes which control tissue patterning, stem cell maintenance, and differentiation during development. However, dysregulation or mutations in HH signaling leads to genomic instability (GI) and various cancers, for example, germline mutation in PTCH1 lead to Gorlin syndrome, a condition where patients develop numerous basal cell carcinomas and rarely rhabdomyosarcoma (RMS). Activating mutations in SMO have also been recognized in sporadic cases of medulloblastoma and SMO is overexpressed in many other cancers. Recently, studies in several human cancers have shown that GLI1 expression is independent from HH ligand and canonical intracellular signaling through PTCH and SMO. In fact, this aberrantly regulated GLI1 has been linked to several non-canonical oncogenic growth signals such as Kirsten rat sarcoma viral oncogene homolog (KRAS), avian myelocytomatosis virus oncogene cellular homolog (C-MYC), transforming growth factor ß (TGFß), wingless-type MMTV integration site family (WNT) and ß-catenin. Recent studies from our lab and other independent studies demonstrate that aberrantly expressed GLI1 influences the integrity of several DNA damage response and repair signals, and if altered, these networks can contribute to GI and impact tumor response to chemo- and radiation therapies. Furthermore, the ineffectiveness of SMO inhibitors in clinical studies argues for the development of GLI1-specific inhibitors in order to develop effective therapeutic modalities to treat these tumors. In this review, we focus on summarizing current understanding of the molecular, biochemical and cellular basis for aberrant GLI1 expression and discuss GLI1-mediated HH signaling on DNA damage responses, carcinogenesis and chemoresistance.
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Sphingolipids play a very crucial role in many diseases and are well-known as signaling mediators in many pathways. Sphingolipids are produced during the de novo process in the ER (endoplasmic reticulum) from the nonsphingolipid precursor and comprise both structural and bioactive lipids. Ceramide is the central core of the sphingolipid pathway, and its production has been observed following various treatments that can induce several different cellular effects including growth arrest, DNA damage, apoptosis, differentiation, and senescence. Ceramides are generally produced through the sphingomyelin hydrolysis and catalyzed by the enzyme sphingomyelinase (SMase) in mammals. Presently, there are many known SMases and they are categorized into three groups acid SMases (aSMases), alkaline SMases (alk-SMASES), and neutral SMases (nSMases). The yeast homolog of mammalians neutral SMases is inositol phosphosphingolipid phospholipase C. Yeasts generally have inositol phosphosphingolipids instead of sphingomyelin, which may act as a homolog of mammalian sphingomyelin. In this review, we shall explain the structure and function of inositol phosphosphingolipid phospholipase C1, its localization inside the cells, mechanisms, and its roles in various cell responses during replication stresses and diseases. This review will also give a new basis for our understanding for the mechanisms and nature of the inositol phosphosphingolipid phospholipase C1/nSMase.
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Fanconi anemia (FA) is a rare genome instability syndrome with progressive bone marrow failure and cancer susceptibility. FANCJ is one of 17 genes mutated in FA-patients, comprises a DNA helicase that is vital for properly maintaining genomic stability and is known to function in the FA-BRCA DNA repair pathway. While exact role(s) of FANCJ in this repair process is yet to be determined, it is known to interact with primary effector FANCD2. However, FANCJ is not required for FANCD2 activation but is important for its ability to fully respond to DNA damage. In this report, we determined that transient depletion of FANCJ adversely affects stability of FANCD2 and its co-regulator FANCI in multiple cell lines. Loss of FANCJ does not significantly alter cell cycle progression or FANCD2 transcription. However, in the absence of FANCJ, the majority of FANCD2 is degraded by both the proteasome and Caspase-3 dependent mechanism. FANCJ is capable of complexing with and stabilizing FANCD2 even in the absence of a functional helicase domain. Furthermore, our data demonstrate that FANCJ is important for FANCD2 stability and proper activation of DNA damage responses to replication blocks induced by hydroxyurea.
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Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Caspasa 3/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Anemia de Fanconi/enzimología , Complejo de la Endopetidasa Proteasomal/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Línea Celular Tumoral , Daño del ADN , Reparación del ADN , Estabilidad de Enzimas , Anemia de Fanconi/tratamiento farmacológico , Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Hidroxiurea/farmacología , Inhibidores de Proteasoma/farmacología , Unión Proteica , Proteolisis , Interferencia de ARN , Factores de Tiempo , Transfección , UbiquitinaciónRESUMEN
Allyl isothiocyanate (AITC), a constituent of many cruciferous vegetables exhibits significant anticancer activities in many cancer models. Our studies provide novel insights into AITC-induced anticancer mechanisms in human A549 and H1299 non-small cell lung cancer (NSCLC) cells. AITC exposure induced replication stress in NSCLC cells as evidenced by γH2AX and FANCD2 foci, ATM/ATR-mediated checkpoint responses and S and G2/M cell cycle arrest. Furthermore, AITC-induced FANCD2 foci displayed co-localization with BrdU foci, indicating stalled or collapsed replication forks in these cells. Although PITC (phenyl isothiocyanate) exhibited concentration-dependent cytotoxic effects, treatment was less effective compared to AITC. Previously, agents that induce cell cycle arrest in S and G2/M phases were shown to sensitize tumor cells to radiation. Similar to these observations, combination therapy involving AITC followed by radiation treatment exhibited increased DDR and cell killing in NSCLC cells compared to single agent treatment. Combination index (CI) analysis revealed synergistic effects at multiple doses of AITC and radiation, resulting in CI values of less than 0.7 at Fa of 0.5 (50% reduction in survival). Collectively, these studies identify an important anticancer mechanism displayed by AITC, and suggest that the combination of AITC and radiation could be an effective therapy for NSCLC.