RESUMO
Mycobacterium smegmatis Nei2 is a monomeric enzyme with AP ß-lyase activity on single-stranded DNA. Expression of Nei2, and its operonic neighbor Lhr (a tetrameric 3'-to-5' helicase), is induced in mycobacteria exposed to DNA damaging agents. Here, we find that nei2 deletion sensitizes M. smegmatis to killing by DNA inter-strand crosslinker trimethylpsoralen but not to crosslinkers mitomycin C and cisplatin. By contrast, deletion of lhr sensitizes to killing by all three crosslinking agents. We report a 1.45 Å crystal structure of recombinant Nei2, which is composed of N and C terminal lobes flanking a central groove suitable for DNA binding. The C lobe includes a tetracysteine zinc complex. Mutational analysis identifies the N-terminal proline residue (Pro2 of the ORF) and Lys51, but not Glu3, as essential for AP lyase activity. We find that Nei2 has 5-hydroxyuracil glycosylase activity on single-stranded DNA that is effaced by alanine mutations of Glu3 and Lys51 but not Pro2. Testing complementation of psoralen sensitivity by expression of wild-type and mutant nei2 alleles in ∆nei2 cells established that AP lyase activity is neither sufficient nor essential for crosslink repair. By contrast, complementation of psoralen sensitivity of ∆lhr cells by mutant lhr alleles depended on Lhr's ATPase/helicase activities and its tetrameric quaternary structure. The lhr-nei2 operon comprises a unique bacterial system to rectify inter-strand crosslinks.IMPORTANCEThe DNA inter-strand crosslinking agents mitomycin C, cisplatin, and psoralen-UVA are used clinically for the treatment of cancers and skin diseases; they have been invaluable in elucidating the pathways of inter-strand crosslink repair in eukaryal systems. Whereas DNA crosslinkers are known to trigger a DNA damage response in bacteria, the roster of bacterial crosslink repair factors is incomplete and likely to vary among taxa. This study implicates the DNA damage-inducible mycobacterial lhr-nei2 gene operon in protecting Mycobacterium smegmatis from killing by inter-strand crosslinkers. Whereas interdicting the activity of the Lhr helicase sensitizes mycobacteria to mitomycin C, cisplatin, and psoralen-UVA, the Nei2 glycosylase functions uniquely in evasion of damage caused by psoralen-UVA.
Assuntos
Reparo do DNA , Ficusina , Mycobacterium smegmatis , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/enzimologia , Mycobacterium smegmatis/metabolismo , Ficusina/química , Ficusina/farmacologia , Ficusina/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Dano ao DNA , Reagentes de Ligações Cruzadas/química , Cristalografia por Raios X , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Mitomicina/farmacologia , Mitomicina/metabolismo , Deleção de GenesRESUMO
The berberine bridge enzyme (BBE)-like flavoproteins have attracted continuous attention for their capability to catalyze various oxidative reactions. Here we demonstrate that MitR, a secreted BBE-like enzyme, functions as a special drug-binding efflux protein evolved from quinone reductase. Moreover, this protein provides self-resistance to its hosts toward the DNA-alkylating agent mitomycin C with a distinctive strategy, featured by independently performing drug binding and efflux.
Assuntos
Mitomicina , NAD(P)H Desidrogenase (Quinona) , Mitomicina/farmacologia , Mitomicina/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , Oxirredutases/metabolismo , Oxirredutases N-Desmetilantes/metabolismoRESUMO
Both the design of molecules that will interact specifically with DNA and the determination of the mechanism of action of this drug on DNA are important as they allow the control of gene expression. In particular, rapid and precise analysis of this type of interaction is a vital element for pharmaceutical studies. In the present study, a novel reduced graphene oxide/ palladium nanoparticles/ poly(2-amino-4-chlorophenol) (rGO/Pd@PACP) nanocomposite was synthesized by chemical process to modify pencil graphite electrode (PGE) surface. Here, the performance of the newly developed nanomaterial-based biosensor for drug-DNA interaction analysis has been demonstrated. For this purpose, it was determined whether this system, which was developed by selecting a drug molecule (Mitomycin C; MC) known to interact with DNA and a drug molecule (Acyclovir; ACY) that does not interact with DNA, performs a reliable/accurate analysis. Here, ACY was used as a negative control. Compared to bare PGE, the rGO/Pd@PACP nanomaterial modified sensor exhibited 17 times higher sensitivity performance in terms of guanine oxidation signal measured by differential pulse voltammetry (DPV). Moreover, the developed nanobiosensor system provided a highly specific determination between the anticancer drug MC and ACY by discrimination the interactions of these drugs with double-stranded DNA (dsDNA). ACY was also preferred in studies for the optimization of the new nanobiosensor developed. ACY was detected in a concentration as low as 0.0513 µM (51.3 nM) (LOD), and limit of quantification (LOQ) was 0.1711 µM with a linear range from 0.1 to 0.5 µM.
Assuntos
Técnicas Biossensoriais , Grafite , Nanopartículas Metálicas , Grafite/química , Mitomicina/metabolismo , Paládio/química , Aciclovir , Técnicas Eletroquímicas , DNA/química , EletrodosRESUMO
BACKGROUND: Recent evidences highlight role of mitochondria in the development of 5-fluorouracil (5-FU)-induced intestinal toxicity. Mitochondria-targeted antioxidants are well-known for their protective effects in mitochondrial oxidative stress- mediated diseases. In the present study, we investigated protective effect of Mito-TEMPO in 5-FU-induced intestinal toxicity. METHODS: Mito-TEMPO (0.1 mg/kg b.w.) was administered intraperitoneally to male BALB/c mice for 7 days, followed by co-administration of 5-FU for next 4 days (intraperitoneal 12 mg/kg b.w.). Protective effect of Mito-TEMPO on intestinal toxicity was assessed in terms of histopathological alterations, modulation in inflammatory markers, apoptotic cell death, expression of 8-OhDG, mitochondrial functional status and oxidative stress. RESULTS: 5-FU administered animals showed altered intestinal histoarchitecture wherein a shortening and atrophy of the villi was observed. The crypts were disorganized and inflammatory cell infiltration was noted. Mito-TEMPO pre-protected animals demonstrated improved histoarchitecture with normalization of villus height, better organized crypts and reduced inflammatory cell infiltration. The inflammatory markers and myeloperoxidase activity were normalized in mito-TEMPO protected group. A significant reduction in intestinal apoptotic cell death and expression of 8-OhDG was also observed in mito-TEMPO group as compared to 5-FU group. Further, mtROS, mtLPO and mitochondrial antioxidant defense status were improved by mito-TEMPO. CONCLUSION: Mito-TEMPO exerted significant protective effect against 5-FU-induced intestinal toxicity. Therefore, it may be used as an adjuvant in 5-FU chemotherapy.
Assuntos
Antioxidantes , Estresse Oxidativo , Camundongos , Animais , Masculino , Antioxidantes/metabolismo , Mitocôndrias , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Mitomicina/metabolismo , Mitomicina/farmacologia , Mitomicina/uso terapêutico , ApoptoseRESUMO
Cancer stem cells (CSCs) are considered to be responsible for tumor recurrence and metastasis. Therefore, clarification of the mechanisms involved in CSC stemness maintenance and cell fate determination would provide a new strategy for cancer therapy. Unregulated cellular energetics has been accepted as one of the hallmarks of cancer cells, but recent studies have revealed that mitochondrial metabolism can also actively determine CSC fate by affecting nuclear stemness gene expression. Herein, from the perspective of mito-nuclear communication, we review recent progress on the influence of mitochondria on CSC potential from four aspects: metabolism, dynamics, mitochondrial homeostasis, and reactive oxygen species (ROS). Video Abstract.
Assuntos
Mitomicina , Recidiva Local de Neoplasia , Humanos , Recidiva Local de Neoplasia/patologia , Diferenciação Celular , Mitomicina/metabolismo , Mitocôndrias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
A viral shunt can occur when phages going through a lytic cycle, including lysogenic phages triggered by inducing agents (e.g. mitomycin C), results in host lysis and the release of cell constituents and virions. The impact of a viral shunt on the carbon, including methane cycle in soil systems is poorly understood. Here, we determined the effects of mitomycin C on the aerobic methanotrophs in a landfill cover soil. To an extent, our results support a mitomycin C-induced viral shunt, as indicated by the significantly higher viral-like particle (VLP) counts relative to bacteria, elevated nutrient concentrations (ammonium, succinate), and initially impaired microbial activities (methane uptake and microbial respiration) after mitomycin C addition. The trend in microbial activities at <2 days largely corresponded to the expression of the pmoA and 16S rRNA genes. Thereafter (>11 days), the active bacterial community composition significantly diverged in the mitomycin C-supplemented incubations, suggesting the differential impact of mitomycin C on the bacterial community. Collectively, we provide insight on the effects of mitomycin C, and potentially a viral shunt, on the bacteria in the soil environment.
Assuntos
Mitomicina , Solo , Oxirredução , Mitomicina/farmacologia , Mitomicina/metabolismo , RNA Ribossômico 16S/genética , Bactérias , Instalações de Eliminação de Resíduos , Metano/metabolismo , Microbiologia do SoloRESUMO
Zonula occludens-1 (ZO-1) is a scaffolding protein of tight junctions, which seal adjacent epithelial cells, that is also expressed in adherens junctions. The distribution pattern of ZO-1 differs among stratified squamous epithelia, including that between skin and oral buccal mucosa. However, the causes for this difference, and the mechanisms underlying ZO-1 spatial regulation, have yet to be elucidated. In this study, we showed that epithelial turnover and proliferation are associated with ZO-1 distribution in squamous epithelia. We tried to verify the regulation of ZO-1 by comparing normal skin and psoriasis, known as inflammatory skin disease with rapid turnover. We as well compared buccal mucosa and oral lichen planus, known as an inflammatory oral disease with a longer turnover interval. The imiquimod (IMQ) mouse model, often used as a psoriasis model, can promote cell proliferation. On the contrary, we peritoneally injected mice mitomycin C, which reduces cell proliferation. We examined whether IMQ and mitomycin C cause changes in the distribution and appearance of ZO-1. Human samples and mouse pharmacological models revealed that slower epithelial turnover/proliferation led to the confinement of ZO-1 to the uppermost part of squamous epithelia. In contrast, ZO-1 was widely distributed under conditions of faster cell turnover/proliferation. Cell culture experiments and mathematical modelling corroborated these ZO-1 distribution patterns. These findings demonstrate that ZO-1 distribution is affected by epithelial cell dynamics.
Assuntos
Carcinoma de Células Escamosas , Psoríase , Camundongos , Animais , Humanos , Junções Íntimas/metabolismo , Mitomicina/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo , Proteína da Zônula de Oclusão-2/metabolismo , Proliferação de Células , Carcinoma de Células Escamosas/metabolismoRESUMO
BACKGROUND: Bioprospecting of actinobacteria isolated from Kubuqi desert, China for antibacterial, antifungal and cytotoxic metabolites production and their structure elucidation. RESULTS: A total of 100 actinobacteria strains were selectively isolated from Kubuqi desert, Inner Mongolia, China. The taxonomic characterization revealed Streptomyces as the predominant genus comprising 37 different species, along with the rare actinobacterial genus Lentzea. The methanolic extracts of 60.8% of strains exhibited potent antimicrobial activities against Staphylococcus aureus, Micrococcus luteus, Bacillus subtilis, Escherichia coli, Salmonella enterica, Saccharomyces cerevisiae and high to mild in vitro cytotoxicity against PC3 (prostate cancer) and A549 (lung carcinoma) cell lines. The metabolomics analysis by TLC, HPLC-UV/vis, HPLC-MS and NMR showed the presence of compounds with molecular weights ranging from 100 to 1000 Da. The scale-up fermentation of the prioritized anti-Gram-negative strain PU-KB10-4 (Streptomyces griseoviridis), yielded three pure compounds including; griseoviridin (1; 42.0 mgL- 1) with 20 fold increased production as compared to previous reports and its crystal structure as monohydrate form is herein reported for the first time, mitomycin C (2; 0.3 mgL- 1) and a new bacterial metabolite 4-hydroxycinnamide (3; 0.59 mgL- 1). CONCLUSIONS: This is the first report of the bioprospecting and exploration of actinobacteria from Kubuqi desert and the metabolite 4-hydroxycinnamide (3) is first time isolated from a bacterial source. This study demonstrated that actinobacteria from Kubuqi desert are a potential source of novel bioactive natural products. Underexplored harsh environments like the Kubuqi desert may harbor a wider diversity of actinobacteria, particularly Streptomyces, which produce unique metabolites and are an intriguing source to develop medicinally valuable natural products.
Assuntos
Actinobacteria , Produtos Biológicos , Streptomyces , Mitomicina/metabolismo , Bioprospecção , Filogenia , Antibacterianos/química , Produtos Biológicos/farmacologiaRESUMO
Purpose: The gold standard for managing postoperative ocular fibrosis in glaucoma surgery is the chemotherapeutic mitomycin C (MMC) despite its association with significant adverse effects. This study compares in vitro the antifibrotic efficacy and cytotoxicity of the small-molecule TGFß1 inhibitor SB-431542 (SB) to MMC. Methods: To measure collagen contraction, human Tenon's capsule fibroblasts (HTCFs) embedded in a three-dimensional collagen lattice were exposed to 0.2 mg/mL MMC or 20 µM SB followed by incubation with 2 ng/mL TGFß1. Total protein extracted from experimentally treated HTCFs underwent immunoblotting for α-smooth muscle actin (α-SMA), matrix metallopeptidase 9 (MMP-9), and EDA splice-variant fibronectin (EDA-FN) expression. Cytotoxicity and cell metabolism were assessed using LIVE/DEAD staining, lactate dehydrogenase (LDH) assay, and methylthiazole tetrazolium (MTT) assay. Results: Collagen lattice contraction in TGFß1-induced HTCFs was significantly lowered by SB and MMC. Pretreatment with SB and MMC significantly lowered protein expression of α-SMA, MMP-9, and EDA-FN in HTCFs relative to TGFß1 alone. HTCF viability in collagen lattices was significantly reduced with MMC pretreatment but not SB pretreatment. MMC-pretreated HTCFs had a significant increase in LDH release after 3 hours and a decrease in MTT activity after 20 minutes, while SB-pretreated HTCFs showed no significant changes via MTT or LDH assay during the same treatment period. Conclusions: SB shows comparable efficacy to MMC in reducing expression of fibrosis-promoting proteins in HTCFs and in vitro scarring activity. SB distinguishes itself from MMC by exhibiting less cytotoxicity in both two-dimensional and three-dimensional in vitro assays. Translational Relevance: This study demonstrates in vitro the potential of SB as a safer alternative ocular antifibrotic agent.
Assuntos
Glaucoma , Mitomicina , Humanos , Mitomicina/metabolismo , Mitomicina/farmacologia , Cápsula de Tenon/metabolismo , Cápsula de Tenon/patologia , Cicatriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/farmacologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Colágeno , Glaucoma/cirurgiaRESUMO
Mitomycin C, (MC), an antitumor drug used in the clinics, is a DNA alkylating agent. Inert in its native form, MC is reduced to reactive mitosenes in cellulo which undergo nucleophilic attack by DNA bases to form monoadducts as well as interstrand crosslinks (ICLs). These properties constitute the molecular basis for the cytotoxic effects of the drug. The mechanism of DNA alkylation by mitomycins has been studied for the past 30â years and, until recently, the consensus was that drugs of the mitomycins family mainly target CpG sequences in DNA. However, that paradigm was recently challenged. Here, we relate the latest research on both MC and dicarbamoylmitomycin C (DMC), a synthetic derivative of MC which has been used to investigate the regioselectivity of mitomycins DNA alkylation as well as the relationship between mitomycins reductive activation pathways and DNA adducts stereochemical configuration. We also review the different synthetic routes to access mitomycins nucleoside adducts and oligonucleotides containing MC/DMC DNA adducts located at a single position. Finally, we briefly describe the DNA structural modifications induced by MC and DMC adducts and how site specifically modified oligonucleotides have been used to elucidate the role each adduct plays in the drugs cytotoxicity.
Assuntos
Adutos de DNA , Mitomicina , Mitomicina/farmacologia , Mitomicina/química , Mitomicina/metabolismo , DNA/química , OligonucleotídeosRESUMO
Hypoglycemia is associated with cognitive dysfunction, but the exact mechanisms have not been elucidated. Our previous study found that severe hypoglycemia could lead to cognitive dysfunction in a type 1 diabetes (T1D) mouse model. Thus, the aim of this study was to further investigate whether the mechanism of severe hypoglycemia leading to cognitive dysfunction is related to oxidative stress-mediated pericyte loss and blood-brain barrier (BBB) leakage. A streptozotocin T1D model (150 mg/kg, one-time intraperitoneal injection), using male C57BL/6J mice, was used to induce hypoglycemia. Brain tissue was extracted to examine for neuronal damage, permeability of BBB was investigated through Evans blue staining and electron microscopy, reactive oxygen species and adenosine triphosphate in brain tissue were assayed, and the functional changes of pericytes were determined. Cognitive function was tested using Morris water maze. Also, an in vitro glucose deprivation model was constructed. The results showed that BBB leakage after hypoglycemia is associated with excessive activation of oxidative stress and mitochondrial dysfunction due to glucose deprivation/reperfusion. Interventions using the mitochondria-targeted antioxidant Mito-TEMPO in both in vivo and in vitro models reduced mitochondrial oxidative stress, decreased pericyte loss and apoptosis, and attenuated BBB leakage and neuronal damage, ultimately leading to improved cognitive function.
Assuntos
Disfunção Cognitiva , Diabetes Mellitus Tipo 1 , Hipoglicemia , Camundongos , Animais , Masculino , Barreira Hematoencefálica/metabolismo , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Pericitos/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Camundongos Endogâmicos C57BL , Mitocôndrias , Glucose/metabolismo , Hipoglicemia/complicações , Hipoglicemia/tratamento farmacológico , Hipoglicemia/metabolismo , Mitomicina/metabolismo , Disfunção Cognitiva/complicações , Disfunção Cognitiva/tratamento farmacológico , Disfunção Cognitiva/metabolismoRESUMO
Purpose: Cytotoxic agents such as mitomycin C (MMC) are part of the mainstay treatment for limiting subconjunctival scarring following glaucoma filtration surgery (GFS). However, a safer antifibrotic therapy is clinically needed. The anti-scarring properties of 3',4'-dihydroxyflavonol (DiOHF) were evaluated in a mouse model of GFS and in cultured human Tenon's fibroblasts (HTFs). Methods: GFS was performed in C57BL/6 mice receiving daily intraperitoneal injections of DiOHF or vehicle or a single intraoperative injection of MMC. Eyes were harvested on day 14 for assessment of collagen deposition, expression of alpha-smooth muscle actin (α-SMA), cluster of differentiation 31 (CD31), and 4-hydroxy-2-nonenal (4HNE) in the conjunctiva/Tenon's layer. The inhibitory effects of DiOHF on transforming growth factor ß (TGFß)-induced responses were also assessed in HTFs. Results: Treatment with DiOHF demonstrated a reduction in collagen deposition at the GFS site compared to vehicle-treated mice. The degree of 4HNE-positive fluorescence was significantly reduced in DiOHF-treated eyes compared to the other groups, indicating a decrease in oxidative stress. A reduction in expression of α-SMA and CD31 was seen in DiOHF-treated conjunctiva compared to those treated with vehicle. Concordant results were demonstrated in cultured HTFs in vitro. Furthermore, treatment of cultured HTFs with DiOHF also displayed a reduction in the proliferation, migration, and contractility of HTFs. Conclusions: Treatment with DiOHF reduces scarring and angiogenesis in the conjunctiva of mice with GFS at a level comparable to MMC. The reduction in oxidative stress suggests that DiOHF may suppress scarring via different mechanisms from MMC. Translational Relevance: DiOHF may be a safer and superior wound modulating agent than conventional antifibrotic therapy in GFS.
Assuntos
Cirurgia Filtrante , Glaucoma , Animais , Colágeno/metabolismo , Colágeno/farmacologia , Modelos Animais de Doenças , Fibroblastos/metabolismo , Flavonóis , Glaucoma/tratamento farmacológico , Glaucoma/cirurgia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mitomicina/metabolismo , Mitomicina/farmacologia , Mitomicina/uso terapêutico , Cápsula de Tenon/metabolismoRESUMO
The pluripotency maintenance of pluripotent stem cells (PSCs) requires the suitable microenvironment, which commonly provided by feeder layers. However, the preparation of feeder layers is time consuming and labor exhaustive, and the feeder cells treated with mitomycin C or γ-ray irradiation bring heterologous contamination. In this study, mouse embryonic fibroblasts (MEFs) were treated by methanol to generate chemical fixed feeder cells, and bovine embryonic stem cells F7 (bESC-F7) cultured on this feeder layer. Then the pluripotency and metabolism of bESC-F7 cultured on methanol-fixed MEFs (MT-MEFs) named MT-F7 was compared with mitomycin C treated MEFs (MC-MEFs). The results showed that bESC-F7 formed alkaline phosphatase positive colonies on MT-MEFs, the relative expression of pluripotent markers of these cells was different from the bESCs cultured on the MC-MEFs (MC-F7). The long-term cultured MT-F7 formed embryoid bodies, showed the ability to differentiate into three germ layers similar to MC-F7. The analyses of RNA-seq data showed that MT-MEFs lead bESCs to novel steady expression patterns of genes regulating pluripotency and metabolism. Furthermore, the bovine expanded pluripotent stem cells (bEPSCs) cultured on MT-MEFs formed classical colonies, maintained pluripotency, and elevated metabolism. In conclusion, MT-MEFs were efficient feeder layer that maintain the distinctive pluripotency and metabolism of PSCs.
Assuntos
Metanol , Células-Tronco Pluripotentes , Animais , Bovinos , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Células Alimentadoras/metabolismo , Fibroblastos , Metanol/metabolismo , Camundongos , Mitomicina/metabolismo , Células-Tronco Pluripotentes/metabolismoRESUMO
BRCA2 is crucial for repairing DNA double-strand breaks with high fidelity, and loss of BRCA2 increases the risks of developing breast and ovarian cancers. Herein, we show that BRCA2 is inactively mutated in 10% of gastric and 7% of colorectal adenocarcinomas, and that this inactivation is significantly correlated with microsatellite instability. Villin-driven Brca2 depletion promotes mouse gastrointestinal tumor formation when genome instability is increased. Whole-genome screening data showed that these BRCA2 monoallelic and biallelic mutant tumors were selectively inhibited by mitomycin C. Mechanistically, mitomycin C provoked double-strand breaks in cancer cells that often recruit wild-type BRCA2 for repair; the failure to repair double-strand breaks caused cell-cycle arrest at the S phase and p53-mediated cell apoptosis of BRCA2 monoallelic and biallelic mutant tumor cells. Our study unveils the role of BRCA2 loss in the development of gastrointestinal tumors and provides a potential therapeutic strategy to eliminate BRCA2 monoallelic and biallelic mutant tumors through mitomycin C.
Assuntos
Proteína BRCA2/deficiência , Neoplasias Gastrointestinais/genética , Mitomicina/metabolismo , Animais , Humanos , CamundongosRESUMO
INTRODUCTION: Mitomycin C (MMC) is commonly used in patients with colorectal peritoneal metastases (CPM) treated with cytoreductive surgery plus hyperthermic intraperitoneal chemotherapy (CRS + HIPEC). MMC requires metabolic activation prior to exert its cytotoxic effect of which the main activating enzymes are NQO1 and POR. However, not all patients are able to activate MMC for example due to polymorphisms in the genes encoding these enzymes. The aim of this study was to investigate the association of NQO1∗2, NQO1∗3, and POR∗28 with the efficacy of CRS + HIPEC with MMC in patients with CPM. METHOD: A retrospective follow-up design was used to study genetic association in patients with histologically proven CPM treated with CRS + HIPEC with MMC with respect to peritoneal recurrence rate after 3 months (primary endpoint), after 6 months, disease-free survival and overall survival. Genetic polymorphisms NQO1∗2, NQO1∗3, and POR∗28 were tested for association. RESULTS: A total of 253 patients were included. In NQO1∗3 carriers the peritoneal recurrence rate 3 and 6 months after HIPEC was significantly higher than in wild type patients, respectively 30.0% vs 3.8% (p = 0.009) and 40.0% vs 12.1% (p = 0.031). In line with these results, NQO1∗3 was associated with a shorter disease-free survival (HR 2.04, 95% CI [1.03-4.03]). There was no significant association with overall survival (HR 1.42, 95% CI [0.66-3.07]). CONCLUSION: Carriership of the NQO1∗3 allele is associated with worse peritoneal recurrence rate and disease-free survival. These results suggest that individualization of patients treated with CRS + HIPEC based upon pharmacogenetics may be beneficial.
Assuntos
Antibióticos Antineoplásicos/uso terapêutico , Carcinoma/terapia , Neoplasias Colorretais/terapia , Sistema Enzimático do Citocromo P-450/genética , Mitomicina/uso terapêutico , NAD(P)H Desidrogenase (Quinona)/genética , Neoplasias Peritoneais/terapia , Variantes Farmacogenômicos/genética , Idoso , Alelos , Antibióticos Antineoplásicos/metabolismo , Carcinoma/secundário , Neoplasias Colorretais/patologia , Sistema Enzimático do Citocromo P-450/metabolismo , Procedimentos Cirúrgicos de Citorredução , Intervalo Livre de Doença , Feminino , Humanos , Quimioterapia Intraperitoneal Hipertérmica , Masculino , Pessoa de Meia-Idade , Mitomicina/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , Neoplasias Peritoneais/secundário , Prognóstico , Estudos RetrospectivosRESUMO
Toxin-antitoxin systems are known to be involved in many bacterial functions that can lead to growth arrest and cell death in response to stress. Typically, toxin and antitoxin genes of type I systems are located in opposite strands, where the antitoxin is a small antisense RNA (sRNA). In the present work we show that the sRNA IsrA from Salmonella Typhimurium down-regulates the expression of its overlapping gene STM0294.1n. Multiple sequence alignment and comparative structure analysis indicated that STM0294.1n belongs to the SymE toxin superfamily, and the gene was renamed iasE (IsrA-overlapping gene with similarity to SymE). The iasE expression was induced in response to mitomycin C, an SOS-inducing agent; conversely, IsrA overexpression repressed the iasE expression even in the presence of mitomycin C. Accordingly, the inactivation of IsrA with an anti-IsrA RNA expressed in trans abrogated the repressive effect of IsrA on the iasE expression. On the other hand, iasE overexpression, as well as the blockage of the antisense IsrA function, negatively affected bacterial growth, arguing for a toxic effect of the iasE gene product. Besides, a bacterial lysate obtained from the iasE-overexpressing strain exhibited endoribonuclease activity, as determined by a fluorometric assay based on fluorescent reporter RNAs. Together, these results indicate that the IasE/IsrA pair of S. Typhimurium constitutes a functional type I toxin-antitoxin system.
Assuntos
Proteínas de Bactérias/genética , RNA Antissenso/genética , RNA Bacteriano/genética , Resposta SOS em Genética/genética , Salmonella typhimurium/genética , Sequência de Aminoácidos , Antitoxinas/genética , Toxinas Bacterianas/genética , Endorribonucleases/metabolismo , Regulação Bacteriana da Expressão Gênica , Mitomicina/metabolismo , Modelos Moleculares , Mutação , Fases de Leitura Aberta/genética , Conformação Proteica , Dobramento de ProteínaRESUMO
The complications from surgery associated peritoneal adhesion can be alleviated by combination of physical isolation and pharmaceutical treatment. This work aims to develop thermo-sensitive hydrogel barrier by combining mitomycin C (MMC) with modified tempo oxidized nanocellulose (cTOCN) through EDC/NHS-chemical conjugation followed by integration with methyl cellulose (MC). The MMC was successfully combined with cTOCN and ensured controlled release of MMC from hydrogel throughout 14 days. Amount of MC (1.5, 2.5, 3.5% w/v) was proportional to gelation time and inversely proportional to degradation of hydrogel. The optimized hydrogel (C2.5T1M0.2) needed only 30 s for thermoreversible sol-gel (4â-37â) phenomenon and did not show in vitro fibroblast cells toxicity as well as ensured complete adhesion prevention efficacy, reperitonealization in rat side wall-cecal abrasion model. Overall, the developed C2.5T1M0.2 thermo-gel advances state-of-the-art in view of cytocompatibility, mechanical stability, optimum degradation, good injectability, sustain drug release from surgical sites, and satisfactory de novo anti-adhesion capacity.
Assuntos
Celulose/química , Hidrogéis/química , Mitomicina/química , Peritônio/patologia , Aderências Teciduais/prevenção & controle , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Força Compressiva , Portadores de Fármacos/química , Liberação Controlada de Fármacos , Hidrogéis/farmacologia , Hidrogéis/uso terapêutico , Camundongos , Mitomicina/metabolismo , Mitomicina/uso terapêutico , Ratos , Ratos Sprague-Dawley , Reologia , Temperatura , ViscosidadeRESUMO
Mesothelin is a protein expressed at high levels on the cell surface in a variety of cancers, with limited expression in healthy tissues. The presence of mesothelin on tumor tissue correlates with increased invasion and metastasis, and resistance to traditional chemotherapies, through mechanisms that remain poorly understood. Molecules that specifically recognize mesothelin and interrupt its contribution to tumor progression have significant potential for targeted therapy and targeted drug delivery applications. A number of mesothelin-targeting therapies are in preclinical and clinical development, although none are currently approved for routine clinical use. In this work, we report the development of a mesothelin-targeting protein based on the fibronectin type-III non-antibody protein scaffold, which offers opportunities for applications where antibodies have limitations. We engineered protein variants that bind mesothelin with high affinity and selectively initiate apoptosis in tumor cells expressing mesothelin. Interestingly, apoptosis does not occur through a caspase-mediated pathway and does not require downregulation of cell-surface mesothelin, suggesting a currently unknown pathway through which mesothelin contributes to cancer progression. Importantly, simultaneous treatment with mesothelin-binding protein and chemotherapeutic mitomycin C had a greater cytotoxic effect on mesothelin-positive cells compared to either molecule alone, underscoring the potential for combination therapy including biologics targeting mesothelin.
Assuntos
Antineoplásicos , Sistemas de Liberação de Medicamentos/métodos , Fibronectinas , Proteínas Ligadas por GPI , Engenharia de Proteínas/métodos , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fibronectinas/química , Fibronectinas/genética , Fibronectinas/metabolismo , Fibronectinas/farmacologia , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/metabolismo , Humanos , Células MCF-7 , Mesotelina , Mitomicina/química , Mitomicina/metabolismo , Mitomicina/farmacologia , Ligação ProteicaRESUMO
PURPOSE: To find safer and more effective drugs than mitomycin C to prevent conjunctival fibrosis in a rabbit model. METHODS: Twenty-four rabbits were involved and randomly divided into four groups. Limbus-based peritomy was performed at the superior cornea, and normal saline (NS group), mitomycin C (MMC group), SR (SR group), or TC (TC group)-coated silicone plate was inserted at the sub-Tenon's space in each group. Conjunctival congestion was evaluated at 1 and 4 weeks postoperatively. At 4 weeks, the numbers of inflammatory cells, fibroblasts, myofibroblasts, blood vessels, and goblet cells were counted in the conjunctiva and Tenon's capsule around the silicone plate. RESULTS: At 4 weeks, conjunctival congestion was significantly less than that observed at 1 week in the SR and TC groups (p < 0.05), whereas the number of myofibroblasts was significantly lower in the MMC and TC groups (p < 0.05). The conjunctiva was significantly less congested in the TC group versus the other groups at 1 week and 4 weeks (p < 0.05). The TC group had the lowest number of inflammatory cells and MMC group had the lowest number of goblet cells among all groups (p < 0.05). CONCLUSIONS: The TC-coated silicone plate was more effective in inhibiting inflammation and fibrosis versus the MMC-coated silicone plate and was associated with fewer adverse effects in the rabbit model.
Assuntos
Doenças da Túnica Conjuntiva/cirurgia , Fibrose/cirurgia , Tacrolimo/farmacologia , Animais , Túnica Conjuntiva/patologia , Túnica Conjuntiva/cirurgia , Doenças da Túnica Conjuntiva/terapia , Córnea/cirurgia , Modelos Animais de Doenças , Fibroblastos/efeitos dos fármacos , Fibrose/terapia , Células Caliciformes , Masculino , Mitomicina/metabolismo , Mitomicina/farmacologia , Coelhos , Silicones , Tacrolimo/metabolismo , Cápsula de Tenon/cirurgiaRESUMO
All organisms possess DNA repair pathways that are used to maintain the integrity of their genetic material. Although many DNA repair pathways are well understood, new pathways continue to be discovered. Here, we report an antibiotic specific DNA repair pathway in Bacillus subtilis that is composed of a previously uncharacterized helicase (mrfA) and exonuclease (mrfB). Deletion of mrfA and mrfB results in sensitivity to the DNA damaging agent mitomycin C, but not to any other type of DNA damage tested. We show that MrfAB function independent of canonical nucleotide excision repair, forming a novel excision repair pathway. We demonstrate that MrfB is a metal-dependent exonuclease and that the N-terminus of MrfB is required for interaction with MrfA. We determined that MrfAB failed to unhook interstrand cross-links in vivo, suggesting that MrfAB are specific to the monoadduct or the intrastrand cross-link. A phylogenetic analysis uncovered MrfAB homologs in diverse bacterial phyla, and cross-complementation indicates that MrfAB function is conserved in closely related species. B. subtilis is a soil dwelling organism and mitomycin C is a natural antibiotic produced by the soil bacterium Streptomyces lavendulae. The specificity of MrfAB suggests that these proteins are an adaptation to environments with mitomycin producing bacteria.