RESUMO
Glipizide; an insulin secretagogue belonging to the sulfonylurea class, is a widely used antidiabetic drug for managing type 2 diabetes. However, the need for life-long administration and repeated doses poses challenges in maintaining optimal blood glucose levels. In this regard, orally active sustained-release nano-formulations can be a better alternative to traditional antidiabetic formulations. The present study explored an innovative approach by formulating orally active sustained-release nano-micelles using the amphiphilic lauric acid-conjugated-F127 (LAF127) block copolymer. LAF127 block copolymer was synthesized through esterification and thoroughly characterized before being employed to develop glipizide-loaded nano-micelles (GNM) via the thin-film hydration technique. The optimized formulation exhibited mean particle size of 341.40 ± 3.21 nm and depicted homogeneous particle size distribution with a polydispersity index (PDI) < 0.2. The formulation revealed a surface charge of -17.11 ± 6.23 mV. The in vitro release studies of glipizide from developed formulation depicted a sustained release profile. Drug loaded micelles exhibited a substantial reduction in blood glucose levels in diabetic rats for a duration of up to 24 h. Notably, neither the blank nano-micelles of LAF127 nor the drug loaded micelles manifested any indications of toxicity in healthy rats. This study provides an insight on suitability of synthesized LAF127 block copolymer for development of effective oral drug delivery systems for anti-diabetic activity without any significant adverse effects.
RESUMO
PURPOSE: Glioblastoma (GBM) is a fatal primary brain tumor with extremely poor clinical outcomes. The anticancer efficiency of tyrosine kinase inhibitors (TKIs) has been shown in GBM and other cancer, with limited therapeutic outcomes. In the current study, we aimed to investigate the clinical impact of active proline-rich tyrosine kinase-2 (PYK2) and epidermal growth factor receptor (EGFR) in GBM and evaluate its druggability by a synthetic TKI-Tyrphostin A9 (TYR A9). METHODS: The expression profile of PYK2 and EGFR in astrocytoma biopsies (n = 48) and GBM cell lines were evaluated through quantitative PCR, western blots, and immunohistochemistry. The clinical association of phospho-PYK2 and EGFR was analyzed with various clinicopathological features and the Kaplan-Meier survival curve. The phospho-PYK2 and EGFR druggability and subsequent anticancer efficacy of TYR A9 was evaluated in GBM cell lines and intracranial C6 glioma model. RESULTS: Our expression data revealed an increased phospho-PYK2, and EGFR expression aggravates astrocytoma malignancy and is associated with patients' poor survival. The mRNA and protein correlation analysis showed a positive association between phospho-PYK2 and EGFR in GBM tissues. The in-vitro studies demonstrated that TYR A9 reduced GBM cell growth, cell migration, and induced apoptosis by attenuating PYK2/EGFR-ERK signaling. The in-vivo data showed TYR A9 treatment dramatically reduced glioma growth with augmented animal survival by repressing PYK2/EGFR-ERK signaling. CONCLUSION: Altogether, this study report that increased phospho-PYK2 and EGFR expression in astrocytoma was associated with poor prognosis. The in-vitro and in-vivo evidence underlined translational implication of TYR A9 by suppressing PYK2/EGFR-ERK modulated signaling pathway. The schematic diagram displayed proof of concept of the current study indicating activated PYK2 either through the Ca2+/Calmodulin-dependent protein kinase II (CAMKII) signaling pathway or autophosphorylation at Tyr402 induces association to the SH2 domain of c-Src that leads to c-Src activation. Activated c-Src in turn activates PYK2 at other tyrosine residues that recruit Grb2/SOS complex and trigger ERK½ activation. Besides, PYK2 interaction with c-Src acts as an upstream of EGFR transactivator that can activate the ERK½ signaling pathway, which induces cell proliferation and cell survival by increasing anti-apoptotic proteins or inhibiting pro-apoptotic proteins. TYR A9 treatment attenuate GBM cell proliferation and migration; and induce GBM cell death by inhibiting PYK2 and EGFR-induced ERK activation.
Assuntos
Astrocitoma , Glioblastoma , Glioma , Animais , Glioblastoma/tratamento farmacológico , Quinase 2 de Adesão Focal/metabolismo , Transdução de Sinais , Receptores ErbB/metabolismo , Fosforilação , Astrocitoma/tratamento farmacológicoRESUMO
The therapeutic benefits of phytochemicals in the treatment of various illnesses and disorders are well documented. They show significant promise for the discovery and creation of novel medications for treating a variety of human diseases. Numerous phytoconstituents have shown antibiotic, antioxidant, and wound-healing effects in the conventional system. Traditional medicines based on alkaloids, phenolics, tannins, saponins, terpenes, steroids, flavonoids, glycosides, and phytosterols have been in use for a long time and are crucial as alternative treatments. These phytochemical elements are crucial for scavenging free radicals, capturing reactive carbonyl species, changing protein glycation sites, inactivating carbohydrate hydrolases, fighting pathological conditions, and accelerating the healing of wounds. In this review, 221 research papers have been reviewed. This research sought to provide an update on the types and methods of formation of methylglyoxal-advanced glycation end products (MGO-AGEs) and molecular pathways induced by AGEs during the progression of the chronic complications of diabetes and associated diseases as well as to discuss the role of phytoconstituents in MGO scavenging and AGEs breaking. The development and commercialization of functional foods using these natural compounds can provide potential health benefits.
Assuntos
Diabetes Mellitus , Aldeído Pirúvico , Humanos , Aldeído Pirúvico/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Óxido de MagnésioRESUMO
The present study illustrates the photosensitizing behavior of mefloquine (MQ) in human skin keratinocytes under ambient doses of UVB and sunlight exposure. Photochemically, MQ generated reactive oxygen species superoxide radical, hydroxyl radical, and singlet oxygen through type I and type II photodynamic reactions, respectively, which caused photooxidative damage to DNA and formed localized DNA lesions cyclobutane pyrimidine dimers. Photosensitized MQ reduced the viability of keratinocytes to 25 %. Significant level of intracellular reactive oxygen species (ROS) generation was estimated through fluorescence probe DCF-H2. Increased apoptotic cells were evident through AO/EB staining and phosphatidyl serine translocation in cell membrane. Single-stranded DNA damage was marked through single-cell gel electrophoresis. Mitochondrial membrane depolarization and lysosomal destabilization were evident. Upregulation of Bax and p21 and downregulation of Bcl-2 genes and corresponding protein levels supported apoptotic cell death of keratinocyte cells. Cyclobutane pyrimidine dimers (CPDs) were confirmed through immunofluorescence. In addition, hallmarks of apoptosis and G2/M phase cell cycle arrest were confirmed through flow cytometry analysis. Our findings suggest that MQ may damage DNA and produce DNA lesions which may induce differential biological responses in the skin on brief exposure to UVB and sunlight.
Assuntos
Apoptose/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Mefloquina/farmacologia , Fármacos Fotossensibilizantes/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Raios Ultravioleta/efeitos adversos , Células Cultivadas , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Queratinócitos/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Pele/efeitos dos fármacos , Pele/metabolismo , Luz Solar/efeitos adversos , Proteína X Associada a bcl-2/metabolismoRESUMO
In the published publication [...].
RESUMO
Pyrimethamine (PYR) is used to treat parasitic infections including toxoplasmosis, pneumonia and cystoisosporiasis in HIV patients. Various oral medicines have shown phototoxicity therefore, we aimed to study the phototoxicity of PYR and its molecular mechanism involving stress responsive lysosomal protein Lamp2 and mitochondrial mediated signaling pathway under normal UVA/B exposure. We found that photodegradation and subsequent photoproduct formation was evident through LCMS/MS analysis. Photosensitized PYR produces ROS that cause damage to DNA, cell membrane and membrane bound organelles in human keratinocytes. PYR triggered cytotoxicity and phototoxicity that was evident through MTT and NRU assay respectively. Intracellular ROS generation caused phosphatidyl serine (PS) translocation in cell membrane, lysosome membrane permeabilization (LMP) and mitochondrial membrane potential (MMP) collapse that was further validated through caspase3 activation. DNA damage was measured as tail DNA formation and cell cycle arrest in G1 phase. Photosensitized PYR induces oxidative stress in the form of overexpression of Lamp2 that ultimately led to cellular apoptosis. Moreover, the effects of UVB were higher than UVA, probably due to its direct interaction with various macromolecules. We propose that photoexcited PYR may be harmful to human health even at normal sunlight exposure. Therefore, protective procedures should be practiced during PYR medication.
Assuntos
Dermatite Fototóxica , Infecções por HIV , Dermatite Fototóxica/etiologia , Dermatite Fototóxica/metabolismo , Humanos , Queratinócitos , Lisossomos , Fosfatidilserinas/metabolismo , Pirimetamina/metabolismo , Pirimetamina/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Luz Solar , Raios Ultravioleta/efeitos adversosRESUMO
Quercetin is one of the most important plant flavanols, having several pharmacological and biological uses. Quercetin (Q) is an extremely hydrophobic phytochemical and has poor intracellular absorption, which makes its use limited. Present research demonstrates that quercetin-loaded PLGA nanoparticles (PLGA-QNPs) could overcome its low hydrophilicity and improve its anti-cancer potential. PLGA nanoparticles loaded with Q were prepared by the solvent evaporation technique and its anticancer activity was examined in vitro as well as in vivo. The cell viability was assessed through MTT assay and apoptosis was assayed through Hoechst-PI and EB/AO double staining followed by mitochondrial damage through Mito-tracker RMX-Ros. Gene expression was examined through RT-PCR. Cell cycle arrest in G2/M phase was analyzed through FACS. The results obtained revealed that PLGA-QNPs significantly reduced the viability of human cervical and breast cancer cell lines. PLGA-QNPs induced apoptosis in human cervical cancer cells in a dose dependent manner. The gene expression of PI3K/AKT was down-regulated and FoxO1 was upregulated in PLGA-QNP-treated cells, which showed a high expression level of active Caspase-3 and 7, which are responsible for apoptosis. In addition, PLGA-QNPs reduced the average number of tumors and prolonged the tumor latency period in DMBA-induced mammary adenocarcinoma SD rats. These findings suggest that PLGA-QNPs inhibit cervical and breast cancer progression via mitochondrial dependent Caspase-3 and 7 and mitochondrial independent FoxO1 activation with concomitant suppression of the PI3K/AKT pathway. For future studies, we suggest that potential druggability efficacy and clinical development of anticancer PLGA-QNPs need to be evaluated intensely for successful anticancer drug development.
RESUMO
PURPOSE: Epithelial to mesenchymal transition (EMT) is pivotal in embryonic development and wound healing, whereas in cancer it inflicts malignancy and drug resistance. The recognition of an EMT-like process in glioma is relatively new and its clinical and therapeutic significance has, as yet, not been fully elucidated. Here, we aimed to delineate the clinical significance of the EMT-like process in glioma and its therapeutic relevance to rabeprazole. METHODS: We investigated the expression profiles of EMT-associated proteins in primary glioma biopsies through Western blotting and immunohistochemistry, and correlated them with various clinicopathological features and data listed in the cancer genome atlas (TCGA). In addition, the anticancer efficacy of rabeprazole and its therapeutic relevance to EMT along with temozolomide chemo-sensitization were assessed using multiple cell-based assays, Western blotting and confocal imaging. For in vivo assessment, we used a stereotaxic C6-rat glioma model. RESULTS: Expression analysis of EMT-associated proteins in glioma biopsies, in conjunction with clinicopathological and TCGA dataset analyses, revealed non-canonical expression of E/N-cadherin and upregulation of GFAP, vimentin and ß-catenin. The increased expression of EMT-associated proteins may attribute to glioma malignancy and a poor patient prognosis. Subsequent in vitro studies revealed that rabeprazole treatment attenuated glioma cell growth and migration, and induced apoptosis. Rabeprazole suppressed EMT by impeding AKT/GSK3ß phosphorylation and/or NF-κB signaling and sensitized temozolomide resistance. Additional in vivo studies showed restricted tumor growth and inhibited expression of EMT-associated proteins after rabeprazole treatment. CONCLUSIONS: Our data revealed (i) a clinical association of the EMT-like process with glioma malignancy and a poor survival and (ii) an anticancer and temozolomide sensitizing effect of rabeprazole by repressing EMT.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Glioma/tratamento farmacológico , Rabeprazol/uso terapêutico , Temozolomida/uso terapêutico , Animais , Antineoplásicos Alquilantes/uso terapêutico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Caderinas/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma/genética , Glioma/metabolismo , Humanos , Estimativa de Kaplan-Meier , Masculino , Ratos Wistar , Vimentina/metabolismoRESUMO
Cervical cancer is common among women with a recurrence rate of 35% despite surgery, radiation, and chemotherapy. Patients receiving chemotherapy or radiotherapy routinely experience several side effects including toxicity, non-targeted damage of tissues, hair loss, neurotoxicity, multidrug resistance (MDR), nausea, anemia and neutropenia. Phytochemicals can interfere with almost every stage of carcinogenesis to prevent cancer development. Many natural compounds are known to activate/deactivate multiple redox-sensitive transcription factors that modulate tumor signaling pathways. Polyphenols have been found to be promising agents against cervical cancer. However, applications of phytochemicals as a therapeutic drug are limited due to low oral bioavailability, poor aqueous solubility and requirement of high doses. Nano-sized phytochemicals (NPCs) are promising anti-cancer agents as they are required in minute quantities which lowers overall treatment costs. Several phytochemicals, including quercetin, lycopene, leutin, curcumin, green tea polyphenols and others have been packaged as nanoparticles and proven to be useful in nano-chemoprevention and nano-chemotherapy. Nanoparticles have high biocompatibility, biodegradability and stability in biological environment. Nano-scale drug delivery systems are excellent source for enhanced drug specificity, improved absorption rates, reduced drug degradation and systemic toxicity. The present review discusses current knowledge in the involvement of phytochemical nanoparticles in cervical cancer therapy over conventional chemotherapy.
Assuntos
Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/uso terapêutico , Nanopartículas/química , Nanopartículas/uso terapêutico , Compostos Fitoquímicos/química , Compostos Fitoquímicos/uso terapêutico , Neoplasias do Colo do Útero/tratamento farmacológico , Antineoplásicos Fitogênicos/administração & dosagem , Disponibilidade Biológica , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Nanopartículas/administração & dosagem , Compostos Fitoquímicos/administração & dosagemRESUMO
Erratic rainfall leading to flash flooding causes huge yield losses in lowland rice. The traditional varieties and landraces of rice possess variable levels of tolerance to submergence stress, but gene discovery and utilization of these resources has been limited to the Sub1A-1 allele from variety FR13A. Therefore, we analysed the allelic sequence variation in three Sub1 genes in a panel of 179 rice genotypes and its association with submergence tolerance. Population structure and diversity analysis based on a 36-plex genome wide genic-SNP assay grouped these genotypes into two major categories representing Indica and Japonica cultivar groups with further sub-groupings into Indica, Aus, Deepwater and Aromatic-Japonica cultivars. Targetted re-sequencing of the Sub1A, Sub1B and Sub1C genes identfied 7, 7 and 38 SNPs making 8, 9 and 67 SNP haplotypes, respectively. Haplotype networks and phylogenic analysis revealed evolution of Sub1B and Sub1A genes by tandem duplication and divergence of the ancestral Sub1C gene in that order. The alleles of Sub1 genes in tolerant reference variety FR13A seem to have evolved most recently. However, no consistent association could be found between the Sub1 allelic variation and submergence tolerance probably due to low minor allele frequencies and presence of exceptions to the known Sub1A-1 association in the genotype panel. We identified 18 cultivars with non-Sub1A-1 source of submergence tolerance which after further mapping and validation in bi-parental populations will be useful for development of superior flood tolerant rice cultivars.
Assuntos
Adaptação Fisiológica/genética , Genes de Plantas , Oryza/genética , Proteínas de Plantas/genética , Alelos , Sequência de Bases , Evolução Molecular , Genótipo , Haplótipos , Fenótipo , Filogenia , Proteínas de Plantas/classificação , Polimorfismo de Nucleotídeo Único , Isoformas de Proteínas/classificação , Isoformas de Proteínas/genética , Alinhamento de SequênciaRESUMO
The Indian initiative, in creating mutant resources for the functional genomics in rice, has been instrumental in the development of 87,000 ethylmethanesulfonate (EMS)-induced mutants, of which 7,000 are in advanced generations. The mutants have been created in the background of Nagina 22, a popular drought- and heat-tolerant upland cultivar. As it is a pregreen revolution cultivar, as many as 573 dwarf mutants identified from this resource could be useful as an alternate source of dwarfing. A total of 541 mutants, including the macromutants and the trait-specific ones, obtained after appropriate screening, are being maintained in the mutant garden. Here, we report on the detailed characterizations of the 541 mutants based on the distinctness, uniformity, and stability (DUS) descriptors at two different locations. About 90% of the mutants were found to be similar to the wild type (WT) with high similarity index (>0.6) at both the locations. All 541 mutants were characterized for chlorophyll and epicuticular wax contents, while a subset of 84 mutants were characterized for their ionomes, namely, phosphorous, silicon, and chloride contents. Genotyping of these mutants with 54 genomewide simple sequence repeat (SSR) markers revealed 93% of the mutants to be either completely identical to WT or nearly identical with just one polymorphic locus. Whole genome resequencing (WGS) of four mutants, which have minimal differences in the SSR fingerprint pattern and DUS characters from the WT, revealed a staggeringly high number of single nucleotide polymorphisms (SNPs) on an average (16,453 per mutant) in the genic sequences. Of these, nearly 50% of the SNPs led to non-synonymous codons, while 30% resulted in synonymous codons. The number of insertions and deletions (InDels) varied from 898 to 2,595, with more than 80% of them being 1-2 bp long. Such a high number of SNPs could pose a serious challenge in identifying gene(s) governing the mutant phenotype by next generation sequencing-based mapping approaches such as Mutmap. From the WGS data of the WT and the mutants, we developed a genic resource of the WT with a novel analysis pipeline. The entire information about this resource along with the panicle architecture of the 493 mutants is made available in a mutant database EMSgardeN22 (http://14.139.229.201/EMSgardeN22).
RESUMO
Rice is a staple cereal of India cultivated in about 43.5Mha area but with relatively low average productivity. Abiotic factors like drought, flood and salinity affect rice production adversely in more than 50% of this area. Breeding rice varieties with inbuilt tolerance to these stresses offers an economically viable and sustainable option to improve rice productivity. Availability of high quality reference genome sequence of rice, knowledge of exact position of genes/QTLs governing tolerance to abiotic stresses and availability of DNA markers linked to these traits has opened up opportunities for breeders to transfer the favorable alleles into widely grown rice varieties through marker-assisted backcross breeding (MABB). A large multi-institutional project, "From QTL to variety: marker-assisted breeding of abiotic stress tolerant rice varieties with major QTLs for drought, submergence and salt tolerance" was initiated in 2010 with funding support from Department of Biotechnology, Government of India, in collaboration with International Rice Research Institute, Philippines. The main focus of this project is to improve rice productivity in the fragile ecosystems of eastern, northeastern and southern part of the country, which bear the brunt of one or the other abiotic stresses frequently. Seven consistent QTLs for grain yield under drought, namely, qDTY1.1, qDTY2.1, qDTY2.2, qDTY3.1, qDTY3.2, qDTY9.1 and qDTY12.1 are being transferred into submergence tolerant versions of three high yielding mega rice varieties, Swarna-Sub1, Samba Mahsuri-Sub1 and IR 64-Sub1. To address the problem of complete submergence due to flash floods in the major river basins, the Sub1 gene is being transferred into ten highly popular locally adapted rice varieties namely, ADT 39, ADT 46, Bahadur, HUR 105, MTU 1075, Pooja, Pratikshya, Rajendra Mahsuri, Ranjit, and Sarjoo 52. Further, to address the problem of soil salinity, Saltol, a major QTL for salt tolerance is being transferred into seven popular locally adapted rice varieties, namely, ADT 45, CR 1009, Gayatri, MTU 1010, PR 114, Pusa 44 and Sarjoo 52. Genotypic background selection is being done after BC2F2 stage using an in-house designed 50K SNP chip on a set of twenty lines for each combination, identified with phenotypic similarity in the field to the recipient parent. Near-isogenic lines with more than 90% similarity to the recipient parent are now in advanced generation field trials. These climate smart varieties are expected to improve rice productivity in the adverse ecologies and contribute to the farmer's livelihood.
Assuntos
Secas , Inundações , Oryza/genética , Locos de Características Quantitativas/genética , Tolerância ao Sal/genética , Adaptação Fisiológica/genética , Mapeamento Cromossômico/métodos , Cromossomos de Plantas/genética , Marcadores Genéticos/genética , Programas Governamentais , Índia , Oryza/crescimento & desenvolvimento , Melhoramento Vegetal/métodos , Polimorfismo de Nucleotídeo Único , Reprodutibilidade dos Testes , Seleção ArtificialRESUMO
Benz(a)anthracene (BA) is an ubiquitous environmental pollutant of polycyclic aromatic hydrocarbon's (PAHs) family. We showed superoxide (O2(-)) catalyzed BA photo modification and apoptosis in HaCaT keratinocytes under sunlight exposure. O2(-) generation was confirmed by quenching through superoxide dismutase (SOD). BA induced photocytotoxicity were investigated through MTT and NRU assay. We proposed DNA insults such as single and double strand breakage and CPDs formation which results in cell cycle arrest and apoptosis by photosensitized BA. BA induced apoptosis was caspase dependent and occurred through a mitochondrial pathway. Reduction of mitochondrial membrane potential, translocation of Bax to mitochondria and cytochrome c release favors involvement of mitochondria in BA phototoxicity. AO/EB double staining and TEM analysis also support apoptotic cell death. We propose a p21 regulated apoptosis via expression of Bax, and cleaved PARP under sunlight exposure. Thus, we conclude that it is imperative to avoid solar radiation during peak hr (between 11A.M. and 3P.M.) when the amount of solar radiation is high, in the light of DNA damage which may lead to mutation or skin cancer through photosensitized BA under sunlight exposure. Concomitantly, investigation is urgently required for the photosafety of BA photoproducts reaching in the environment through photomodification.
Assuntos
Apoptose/efeitos dos fármacos , Benzo(a)Antracenos/toxicidade , Dano ao DNA/efeitos dos fármacos , Mitocôndrias/metabolismo , Superóxidos/química , Apoptose/efeitos da radiação , Benzo(a)Antracenos/análise , Benzo(a)Antracenos/química , Catálise , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA/efeitos da radiação , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Luz , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Superóxidos/metabolismo , Raios Ultravioleta , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Anthrone a tricyclic aromatic hydrocarbon which is toxic environmental pollutant comes in the environment through photooxidation of anthracene. We have studied the photomodification of anthrone under environmental conditions. Anthrone generates reactive oxygen species (ROS) like (1)O2 through Type-II photodynamic reaction. Significant intracellular ROS generation was measured through dichlorohydrofluorescein fluorescence intensity. The generation of (1)O2 was further substantiated by using specific quencher like sodium azide. UV induced photodegradation of 2-deoxyguanosine and photoperoxidation of linoleic acid accorded the involvement of (1)O2 in the manifestation of anthrone phototoxicity. Phototoxicity of anthrone was done on human keratinocytes (HaCaT) through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and neutral red uptake assays. Anthrone induced cell cycle arrest (G2/M-phase) and DNA damage in a concentration dependent manner. We found apoptosis as a pattern of cell death which was confirmed through sub-G1 fraction, morphological changes, caspase-3 activation, acridine orange/ethidium bromide staining and phosphatidylserine translocation. Mitochondrial depolarization and lysosomal destabilization was parallel to apoptotic process. Our RT-PCR results strongly supports our view point of apoptotic cell death through up-regulation of pro-apoptotic genes p21 and Bax, and down regulation of anti-apoptotic gene Bcl2. Therefore, much attention should be paid to concomitant exposure of anthrone and UV-R for its total environmental impact.
Assuntos
Antracenos/efeitos da radiação , Antracenos/toxicidade , Poluentes Ambientais/efeitos da radiação , Poluentes Ambientais/toxicidade , Raios Ultravioleta , Apoptose , Caspase 3/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21/genética , Dano ao DNA , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Ácido Linoleico/química , Ácido Linoleico/efeitos da radiação , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Potencial da Membrana Mitocondrial , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosfatidilserinas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Espécies Reativas de Oxigênio/metabolismo , Oxigênio Singlete/química , Proteína X Associada a bcl-2/genéticaRESUMO
This study aimed to analyze the phototoxic mechanism and photostability of quinine in human skin cell line A375 under ambient intensities of UVA (320-400 nm). Photosensitized quinine produced a photoproduct 6-methoxy-quinoline-4-ylmethyl-oxonium identified through LC-MS/MS. Generation of (1)O2, O2(â¢-), and (â¢)OH was measured and further substantiated through their respective quenchers. Photosensitized Quinine (Q) caused degradation of 2-deoxyguanosine, the most sensitive nucleotide to UV radiation. The intracellular ROS was increased in a concentration-dependent manner. Significant reduction in metabolic status measured in terms of cell viability (54%) at 25 µg mL(-1) was observed through MTT assay. Results of MTT assay accord NRU assay. Single strand DNA breaks and apoptosis were increased significantly (P < 0.01) as observed through comet assay and EB/AO double staining. Photosensitized quinine caused cells to arrest in G2 phase of cell cycle and induced apoptosis (5.08%) as revealed through FACS. Real-Time PCR showed upregulation of p21 (4.56 folds) and p53 (2.811 folds) genes expression. Thus, our study suggests that generation of reactive oxygen species by quinine under ambient intensity of UVA may result into deleterious phototoxic effects among human population.
Assuntos
Apoptose , Inibidor de Quinase Dependente de Ciclina p21/genética , Quinina/farmacologia , Pele/metabolismo , Proteína Supressora de Tumor p53/genética , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Ensaio Cometa , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Quebras de DNA de Cadeia Simples/efeitos dos fármacos , Quebras de DNA de Cadeia Simples/efeitos da radiação , Humanos , Radical Hidroxila/metabolismo , Melanoma , Necrose , Espécies Reativas de Oxigênio/metabolismo , Pele/efeitos dos fármacos , Pele/patologia , Pele/efeitos da radiação , Neoplasias Cutâneas , Superóxidos/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Raios Ultravioleta , Regulação para CimaRESUMO
Ketoprofen (KP) is a widely used nonsteroidal anti-inflammatory drug for the treatment of osteoarthritis and various rheumatic diseases. Currently, KP is applied topically on skin as gel to treat symptoms of pain and inflammation. We have studied the photomodification of KP under natural environmental conditions. KP generates reactive oxygen species (ROS) like ¹O2 through Type-II photodynamic reaction. ¹O2 mediated 2'-dGuO photodegradation, single and double strand breakage were significantly induced by photosensitized KP under sunlight/UV-R exposure. Significant intracellular ROS generation was measured through DCF-DA fluorescence. Linoleic acid photoperoxidation and role of ¹O2 were substantiated by using specific quencher like sodium azide. KP induced cell cycle arrest in G2/M phase and cell death through MTT assay. We found apoptosis as the pattern of cell death which was confirmed through caspase-3 activation, cytochrome-c release from mitochondria, up-regulation of Bax protein and phosphatidylserine translocation. Our RT-PCR result strongly supports our view point of apoptotic cell death through up-regulation of p21 and pro-apoptotic Bax genes expression. Mitochondrial depolarization and lysosomal destabilization were also parallel to apoptotic process. Therefore, much attention should be paid to the topical application of KP and sunlight exposure in the light of skin related photosensitivity and cancers.
Assuntos
Dano ao DNA/fisiologia , Dermatite Fototóxica/metabolismo , Cetoprofeno/toxicidade , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Oxigênio Singlete/metabolismo , Anti-Inflamatórios não Esteroides/toxicidade , Dano ao DNA/efeitos dos fármacos , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/efeitos da radiação , Lisossomos/efeitos dos fármacos , Lisossomos/efeitos da radiação , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/efeitos da radiação , Oxigênio Singlete/efeitos da radiação , Luz Solar/efeitos adversos , Raios Ultravioleta/efeitos adversosRESUMO
Novel trioxane 97/78, developed by Central Drug Research Institute (CDRI), Lucknow has shown promising antimalarial activity. Clinical experience of anti-malarial drugs registered the occurrence of phototoxicity in patients exposed with sunlight subsequent to medication. Photodegradation study has identified one photo-product up to 4h under UV-B/Sunlight by LC-MS/MS. UV-B irradiated 97/78 compound produced ¹O2 via type-II dependent reaction mechanism, corroborated by its specific quencher. 2'-dGuO degradation and % tail development in photochemical as well as comet test, advocated the genotoxic potential of 97/78. The photocytotoxicity assays (MTT and NRU) on HaCaT cell line revealed the considerable decline in cell viability by 97/78. Cell cycle and Annexin V/PI double stain along with AO/EB demonstrated the G2/M phase arrest and apoptosis. Significant caspase-3 activity was measured in photoexcited 97/78 by colorimetric assay. Fluorescence stain with AO/JC-1 confirmed the lysosomal disruption and mitochondrial membrane destabilization by UV-B irradiated 97/78. Gene expression by RT-PCR showed significant upregulation of p21 and pro-apoptotic Bax, but no change observed in Bcl-2. In conclusion, the study highlights ROS mediated DNA damage, lysosomal and mitochondrial destabilization via upregulation of Bax and activation of caspase-3 which further leads to apoptosis.