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1.
iScience ; 26(6): 106896, 2023 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-37332597

RESUMO

Hidradenitis suppurativa (HS) is a skin disorder that causes chronic painful inflammation and hyperproliferation, often with the comorbidity of invasive keratoacanthoma (KA). Our research, employing high-resolution immunofluorescence and data science approaches together with confirmatory molecular analysis, has identified that the 5'-cap-dependent protein translation regulatory complex eIF4F is a key factor in the development of HS and is responsible for regulating follicular hyperproliferation. Specifically, eIF4F translational targets, Cyclin D1 and c-MYC, orchestrate the development of HS-associated KA. Although eIF4F and p-eIF4E are contiguous throughout HS lesions, Cyclin D1 and c-MYC have unique spatial localization and functions. The keratin-filled crater of KA is formed by nuclear c-MYC-induced differentiation of epithelial cells, whereas the co-localization of c-MYC and Cyclin D1 provides oncogenic transformation by activating RAS, PI3K, and ERK pathways. In sum, we have revealed a novel mechanism underlying HS pathogenesis of follicular hyperproliferation and the development of HS-associated invasive KA.

2.
J Med Chem ; 65(21): 14409-14423, 2022 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-36318154

RESUMO

Compound 1 is a potent rexinoid that is highly effective in cancer chemoprevention but elevates serum triglycerides. In an effort to separate the lipid toxicity from the anticancer activity of 1, we synthesized four new analogs of rexinoid 1, of which three rexinoids did not elevate serum triglycerides. Rexinoids 3 and 4 are twice as potent as rexinoid 1 in binding to Retinoid X receptor (RXR). All-trans retinoic acid (ATRA) plays a key role in maintaining skin homeostasis, and rexinoids 3-6 are highly effective in upregulating the genes responsible for the biosynthesis of ATRA. Inflammation plays a key role in skin cancer, and rexinoids 3 and 4 are highly effective in diminishing LPS-induced inflammation. Rexinoids 3 and 4 are highly effective in preventing UVB-induced nonmelanoma skin cancer (NMSC) without displaying any overt toxicities. Biophysical studies of rexinoids 3 and 5 bound to hRXRα-ligand binding domain (LBD) reveal important conformational and dynamical differences in the ligand binding domain.


Assuntos
Neoplasias Cutâneas , Tetra-Hidronaftalenos , Humanos , Tetra-Hidronaftalenos/química , Ligantes , Receptores X de Retinoides/metabolismo , Tretinoína/química , Tretinoína/metabolismo , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/prevenção & controle , Inflamação/tratamento farmacológico , Inflamação/prevenção & controle , Triglicerídeos
3.
Curr Top Med Chem ; 21(10): 908-919, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33902420

RESUMO

BACKGROUND: In the present study, we have explored the utility of QSAR modelling, in silico ADMET, docking, chemical semi-synthesis, and in vitro evaluation studies for the identification of active camptothecin (CPT) derivatives against cancer-targeting human liver (HepG2) and lung (A549) cancer cell lines. METHODS: Two QSAR models were developed as screenings tools using the multiple linear regression (MLR) method followed by ADMET and docking studies. The regression coefficient (r2) and cross-validation regression coefficients (rCV2T) of the QSAR model for the HepG2 cell line was 0.95 and 0.90, respectively, and for the A549 cell line, it was 0.93 and 0.81, respectively. RESULTS: In silico studies show that CPT derivatives (CPT-1 and CPT-6) possess drug-like properties. Docking performed on DNA Topoisomerase-I showed significant binding affinity. Finally, predicted active derivatives were chemically semi synthesized, spectroscopically characterized, and evaluated in-vitro for cytotoxic/anticancer activity against HepG2 and A549 cell lines. CONCLUSION: The experimental results are consistent with the predicted results. These findings may be of immense importance in the anticancer drug development from an inexpensive and widely available natural product, camptothecin.


Assuntos
Antineoplásicos/isolamento & purificação , Produtos Biológicos/isolamento & purificação , Camptotecina/isolamento & purificação , Magnoliopsida/química , Extratos Vegetais/isolamento & purificação , Células A549 , Antineoplásicos/farmacologia , Produtos Biológicos/farmacologia , Camptotecina/farmacologia , DNA Topoisomerases Tipo I/química , Ensaios de Seleção de Medicamentos Antitumorais , Células Hep G2 , Humanos , Modelos Lineares , Simulação de Acoplamento Molecular/métodos , Extratos Vegetais/farmacologia , Ligação Proteica , Relação Quantitativa Estrutura-Atividade , Transdução de Sinais
4.
Am J Physiol Renal Physiol ; 315(1): F45-F56, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29092846

RESUMO

Elevated levels of brain-derived neurotrophic factor (BDNF) in urine of overactive bladder (OAB) patients support the association of BDNF with OAB symptoms, but the causality is not known. Here, we investigated the functionality of BDNF overexpression in rat bladder following bladder wall transfection of either BDNF or luciferase (luciferase) transgenes (10 µg). One week after transfection, BDNF overexpression in bladder tissue and elevation of urine BDNF levels were observed together with increased transcript of BDNF, its cognate receptors (TrkB and p75NTR), and downstream PLCγ isoforms in bladder. BDNF overexpression can induce the bladder overactivity (BO) phenotype which is demonstrated by the increased voiding pressure and reduced intercontractile interval during transurethral open cystometry under urethane anesthesia. A role for BDNF-mediated enhancement of prejunctional cholinergic transmission in BO is supported by the significant increase in the atropine- and neostigmine-sensitive component of nerve-evoked contractions and upregulation of choline acetyltransferase, vesicular acetylcholine transporter, and transporter Oct2 and -α1 receptors. In addition, higher expression of transient receptor channels (TRPV1 and TRPA1) and pannexin-1 channels in conjunction with elevation of ATP and neurotrophins in bladder and also in L6/S1 dorsal root ganglia together support a role for sensitized afferent nerve terminals in BO. Overall, genomic changes in efferent and afferent neurons of bladder induced by the overexpression of BDNF per se establish a mechanistic link between elevated BDNF levels in urine and dysfunctional voiding observed in animal models and in OAB patients.


Assuntos
Trifosfato de Adenosina/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Fibras Colinérgicas/metabolismo , Bexiga Urinária Hiperativa/metabolismo , Bexiga Urinária/inervação , Bexiga Urinária/metabolismo , Urodinâmica , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Modelos Animais de Doenças , Feminino , Proteínas do Tecido Nervoso , Fosfolipase C gama/metabolismo , Pressão , Ratos Sprague-Dawley , Receptor trkB/metabolismo , Receptores de Fatores de Crescimento , Receptores de Fator de Crescimento Neural/metabolismo , Receptores Purinérgicos/metabolismo , Transmissão Sináptica , Transfecção , Regulação para Cima , Bexiga Urinária Hiperativa/genética , Bexiga Urinária Hiperativa/fisiopatologia
5.
Arch Toxicol ; 89(2): 243-58, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24825450

RESUMO

Inhibition mechanism(s) of protein kinase B/Akt1 and its consequences on related cell signaling were investigated in human neuroblastoma SH-SY5Y cells exposed to 4-hydroxy-trans-2-nonenal (4-HNE), one of the most reactive aldehyde by-products of lipid peroxidation. In silico data indicate that 4-HNE interacts with kinase domain of Akt1 with the total docking score of 6.0577 and also forms H-bond to Glu234 residue similar to highly potent Akt1 inhibitor imidazopiperidine analog 8b, in which the protonated imidazole nitrogen involves in two hydrogen bonds between Glu234 and Asp292. The strong hydrogen bonding with Glu234 and hydrophobic interactions with several residues, namely Leu156, Gly157, Val164, Ala177, Tyr229, Ala230, Met281 and Thr291, at the vicinity which is normally occupied by the ribose of ATP, appear to be the main causes of Akt1 inhibition and lead to the significant conformational change on this region of protein. Results of mutational docking prove that Glu234 plays a major role in 4-HNE-mediated Akt1 inhibition. In silico data on Akt inhibition were further validated by observing the down-regulated levels of phosphorylated (Thr308/Ser493) Akt1 as well as the altered levels of the downstream targets of pAkt, namely downregulated levels of pGSK3ß (Ser9), ß-catenin, Bcl2 and upregulated levels of pro-apoptotic markers, namely Bad, Bax, P(53) and caspase-9/3. The cellular fate of such pAkt inhibition was evidenced by increased reactive oxygen species, degraded nuclei, transferase dUTP nick end labeling positive cells and upregulated levels of pJNK1/2. We identified that 4-HNE-mediated Akt1 inhibition was due to the competitive inhibition of ATP by 4-HNE at the kinase domain of ATP binding sites.


Assuntos
Trifosfato de Adenosina/metabolismo , Aldeídos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Sítios de Ligação , Linhagem Celular Tumoral , Humanos , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo
6.
Int J Urol ; 21 Suppl 1: 18-25, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24807488

RESUMO

Bladder pain syndrome/interstitial cystitis is a disease with lower urinary tract symptoms, such as bladder pain and urinary frequency, which results in seriously impaired quality of life of patients. The extreme pain and urinary frequency are often difficult to treat. Although the etiology of bladder pain syndrome/interstitial cystitis is still not known, there is increasing evidence showing that afferent hyperexcitability as a result of neurogenic bladder inflammation and urothelial dysfunction is important to the pathophysiological basis of symptom development. Further investigation of the pathophysiology will lead to the effective treatment of patients with bladder pain syndrome/interstitial cystitis.


Assuntos
Vias Aferentes , Cistite Intersticial , Inflamação , Plasticidade Neuronal , Fármacos do Sistema Sensorial/farmacologia , Vias Aferentes/efeitos dos fármacos , Vias Aferentes/metabolismo , Vias Aferentes/fisiopatologia , Anti-Inflamatórios não Esteroides/farmacologia , Cistite Intersticial/etiologia , Cistite Intersticial/metabolismo , Cistite Intersticial/fisiopatologia , Cistite Intersticial/psicologia , Citocinas/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/fisiopatologia , Sintomas do Trato Urinário Inferior/fisiopatologia , Fator de Crescimento Neural/metabolismo , Qualidade de Vida , Terapias em Estudo , Uretra/inervação , Uretra/fisiopatologia , Bexiga Urinária/inervação , Bexiga Urinária/fisiopatologia
7.
PLoS One ; 9(3): e91946, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24663500

RESUMO

The expression and metabolic profile of cytochrome P450s (CYPs) is largely missing in human brain due to non-availability of brain tissue. We attempted to address the issue by using human brain neuronal (SH-SY5Y) and glial (U373-MG) cells. The expression and activity of CYP1A1, 2B6 and 2E1 were carried out in the cells exposed to CYP inducers viz., 3-methylcholanthrene (3-MC), cyclophosphamide (CPA), ethanol and known neurotoxicant- monocrotophos (MCP), a widely used organophosphorous pesticide. Both the cells show significant induction in the expression and CYP-specific activity against classical inducers and MCP. The induction level of CYPs was comparatively lower in MCP exposed cells than cells exposed to classical inducers. Pre-exposure (12 h) of cells to classical inducers significantly added the MCP induced CYPs expression and activity. The findings were concurrent with protein ligand docking studies, which show a significant modulatory capacity of MCP by strong interaction with CYP regulators-CAR, PXR and AHR. Similarly, the known CYP inducers- 3-MC, CPA and ethanol have also shown significantly high docking scores with all the three studied CYP regulators. The expression of CYPs in neuronal and glial cells has suggested their possible association with the endogenous physiology of the brain. The findings also suggest the xenobiotic metabolizing capabilities of these cells against MCP, if received a pre-sensitization to trigger the xenobiotic metabolizing machinery. MCP induced CYP-specific activity in neuronal cells could help in explaining its effect on neurotransmission, as these CYPs are known to involve in the synthesis/transport of the neurotransmitters. The induction of CYPs in glial cells is also of significance as these cells are thought to be involved in protecting the neurons from environmental insults and safeguard them from toxicity. The data provide better understanding of the metabolizing capability of the human brain cells against xenobiotics.


Assuntos
Encéfalo/citologia , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Monocrotofós/metabolismo , Monocrotofós/toxicidade , Biocatálise/efeitos dos fármacos , Linhagem Celular Tumoral , Sistema Enzimático do Citocromo P-450/química , Humanos , Simulação de Acoplamento Molecular , Neurotoxinas/metabolismo , Neurotoxinas/toxicidade , Conformação Proteica , Transcrição Gênica/efeitos dos fármacos , Xenobióticos/metabolismo , Xenobióticos/toxicidade
8.
ISRN Pharmacol ; 2014: 601653, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24527221

RESUMO

Purpose. The following review focuses on the recent advancements in intravesical drug delivery, which brings added benefit to the therapy of detrusor overactivity and interstitial cystitis/painful bladder syndrome (IC/PBS). Results. Intravesical route is a preferred route of administration for restricting the action of extremely potent drugs like DMSO for patients of interstitial cystitis/painful bladder syndrome (IC/PBS) and botulinum toxin for detrusor overactivity. Patients who are either refractory to oral treatment or need to mitigate the adverse effects encountered with conventional routes of administration also chose this route. Its usefulness in some cases can be limited by vehicle (carrier) toxicity or short duration of action. Efforts have been underway to overcome these limitations by developing liposome platform for intravesical delivery of biotechnological products including antisense oligonucleotides. Conclusions. Adoption of forward-thinking approaches can achieve advancements in drug delivery systems targeted to future improvement in pharmacotherapy of bladder diseases. Latest developments in the field of nanotechnology can bring this mode of therapy from second line of treatment for refractory cases to the forefront of disease management.

9.
Neuromolecular Med ; 15(3): 570-92, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23846855

RESUMO

Developing neurons, derived from the human umbilical cord blood stem cells (hUCBSCs), were investigated for their stage-specific responses against 3-methylcholanthrene (MC), a well-known polycyclic aromatic hydrocarbon. Three-dimensional (3D) molecular docking demonstrates the strong hydrogen bonding and hydrophobic interactions of MC with amino acids of aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT) within 4 Å and subsequent inhibition of cAMP response element-binding protein (CREB), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors. Protein-protein docking also confirms that induced levels of AHR inhibit the neurogenesis-related transcription factor (CREB) with maximum docking scores. In concurrence with in silico data, MC exposure significantly up regulates the expression and activity of AHR, CYP1A1 and glutathione S-transferase P1-1 (GSTP1-1) and down regulates the expression of CREB, AMPA and NMDA receptors in hUCBSC-derived neuronal cells at various maturity (0, 2, 4, 8 days of differentiation). MC-mediated significant down regulation in the expression of stage-specific neuronal markers (Nestin, neural cell adhesion molecule-NCAM, synaptophysin-SYP, CREB, AMPA and N-methyl-D-aspartate receptor subunit 2A-NR2A) was also noticed in cells all through the differentiation. Data identify the possible interference of MC in neuronal transmission and neurogenesis.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células-Tronco Hematopoéticas/efeitos dos fármacos , Metilcolantreno/toxicidade , Células-Tronco Neurais/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/metabolismo , Antígenos CD34/análise , Translocador Nuclear Receptor Aril Hidrocarboneto/química , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sítios de Ligação , Células Cultivadas , Simulação por Computador , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Sangue Fetal/citologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Glutationa S-Transferase pi/genética , Glutationa S-Transferase pi/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Metilcolantreno/química , Metilcolantreno/metabolismo , Microssomos/enzimologia , Simulação de Acoplamento Molecular , Células-Tronco Neurais/metabolismo , Neurogênese/efeitos dos fármacos , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , Receptores de AMPA/metabolismo , Receptores de Hidrocarboneto Arílico/química , Receptores de Hidrocarboneto Arílico/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Antígenos Thy-1/análise
10.
ACS Chem Neurosci ; 4(2): 285-94, 2013 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-23421680

RESUMO

An in vitro model of ischemic cerebral stroke [oxygen-glucose deprivation (OGD) for 6 h followed by 24 h reoxygenation (R)] with PC12 cells increases Ca(2+) influx by upregulating native L-type Ca(2+) channels and reactive oxygen species (ROS) generation. This reactive oxygen species generation and increase in intracellular Ca(2+) triggers the expression of hypoxic homeostasis transcription factors such as hypoxia induced factor-1 alpha (HIF-1α), Cav-beta 3 (Cav ß3), signal transducer and activator of transcription 3 (STAT3), heat shock protein 27 (hsp-27), and cationic channel transient receptor potential melastatin 7 (TRPM7). OGD insulted PC12 cells were subjected to biologically safe doses (5, 10, and 25 µM) of trans-resveratrol in three different treatment groups: 24 h prior to OGD (pre-treatment); 24 h post OGD (post-treatment); and from 24 h before OGD to end of reoxygenation period (whole-treatment). Here, we demonstrated that OGD-R-induced neuronal injury/death is by reactive oxygen species generation, increase in intracellular calcium levels, and decrease in antioxidant defense enzymes. trans-Resveratrol increases the viability of OGD-R insulted PC12 cells, which was assessed by using MTT, NRU, and LDH release assay. In addition, trans-resveratrol significantly decreases reactive oxygen species generation, intracellular Ca(2+) levels, and hypoxia associated transcription factors and also increases the level of antioxidant defense enzymes. Our data shows that the whole-treatment group of trans-resveratrol is most efficient in decreasing hypoxia induced cell death through its antioxidant properties.


Assuntos
Antioxidantes/farmacologia , Cálcio/metabolismo , Hipóxia/metabolismo , Isquemia/metabolismo , Estilbenos/farmacologia , Animais , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Morte Celular , Hipóxia Celular , Proteínas de Choque Térmico HSP27/efeitos dos fármacos , Proteínas de Choque Térmico HSP27/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , L-Lactato Desidrogenase/efeitos dos fármacos , L-Lactato Desidrogenase/metabolismo , Células PC12 , Ratos , Espécies Reativas de Oxigênio/metabolismo , Resveratrol , Fator de Transcrição STAT3/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismo , Superóxido Dismutase/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1 , Canais de Cátion TRPM/efeitos dos fármacos , Canais de Cátion TRPM/metabolismo
11.
Stem Cells Dev ; 22(2): 224-38, 2013 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-22897592

RESUMO

Inhibition mechanisms of protein kinase B (Pkb)/Akt and its consequences on related cell signaling were investigated in human umbilical cord blood stem cells (hUCBSCs) exposed to monocrotophos (MCP, an organophosphate pesticide). In silico data reveal that MCP interacts with kinase and c-terminal regulatory domains of Akt1, resulting into a total docking score of 5.2748 and also forms H-bond between its N-H and Thr-291 residue of Akt1, in addition to possessing several hydrophobic interactions. The main cause of Akt inhibition is considered to be the strong hydrogen bond between N-H and Thr-291, and hydrophobic interactions at Glu-234, and Asp-292 in the vicinity, which is usually occupied by the ribose of ATP, and interaction with residue Phe-161, thus leading to a significant conformational change in that particular portion of the protein. In silico data on Akt inhibition were confirmed by examining the downregulation of phosphorylated (Thr308/Ser493) Akt1 in MCP-exposed hUCBSCs. MCP-mediated altered levels of pAkt downstream targets viz., downregulated pGSK3ß (Ser9), unchanged GSK3αß, and upregulated levels of Bad, P(53), and caspase-9 further confirm the inhibition of pAkt. The cellular fate of such pAkt inhibition was confirmed by increased terminal deoxynucleotide transferase dUTP nick-end labeling positive cells, reduced mitochondrial membrane potential, and the activation of various MAPKs, proapoptotic markers-Bax, and caspases-9/3. Our data demonstrate that Akt1 plays a key role in MCP-induced apoptosis in hUCBSCs. We also identified that such cellular responses of human cord blood stem cells against MCP were due to strong binding and inhibition of kinase and AGC-Kinase-C terminal regulatory domains of Akt1.


Assuntos
Apoptose , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Monocrotofós/efeitos adversos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Via de Sinalização Wnt , Western Blotting , Camptotecina/efeitos adversos , Caspase 3/genética , Caspase 3/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ativação Enzimática , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Células-Tronco Hematopoéticas/patologia , Humanos , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Estresse Oxidativo , Praguicidas/efeitos adversos , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Espécies Reativas de Oxigênio/metabolismo , Transcrição Gênica , beta Catenina/genética , beta Catenina/metabolismo
12.
Toxicol Sci ; 129(2): 392-410, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22733800

RESUMO

The status of xenobiotic metabolism in developing human brain cells is not known. The reason is nonavailability of developing human fetal brain. We investigate the applicability of the plasticity potential of human umbilical cord blood stem cells for the purpose. Characterized hematopoietic stem cells are converted into neuronal subtypes in eight days. The expression and substrate-specific catalytic activity of the cytochrome P450s (CYPs) CYP1A1 and 3A4 increased gradually till day 8 of differentiation, whereas CYP2B6 and CYP2E1 showed highest expression and activity at day 4. There was no significant increase in the expression of CYP regulators, namely, aryl hydrocarbon receptor (AHR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), and glutathione-S-transferase (GSTP1-1) during differentiation. Differentiating cells showed significant induction in the expression of CYP1A1, 2B6, 2E1, 3A4, AHR, CAR, PXR, and GSTP1-1 when exposed to rifampin, a known universal inducer of CYPs. The xenobiotic-metabolizing capabilities of these differentiating cells were confirmed by exposing them to the organophosphate pesticide monocrotophos (MCP), a known developmental neurotoxicant, in the presence and absence of a universal inhibitor of CYPs-cimetidine. Early-differentiating cells (day 2) were found to be more vulnerable to xenobiotics than mature well-differentiated cells. For the first time, we report significant expression and catalytic activity of selected CYPs in human cord blood hematopoietic stem cell-derived neuronal cells at various stages of maturity. We also confirm significant induction in the expression and catalytic activity of selected CYPs in human cord blood stem cell-derived differentiating neuronal cells exposed to known CYP inducers and MCP.


Assuntos
Antígenos CD34/imunologia , Diferenciação Celular , Sistema Enzimático do Citocromo P-450/metabolismo , Sangue Fetal/citologia , Neurônios/citologia , Células-Tronco/enzimologia , Sequência de Bases , Sistema Enzimático do Citocromo P-450/biossíntese , Primers do DNA , Indução Enzimática , Sangue Fetal/enzimologia , Humanos , Neurônios/enzimologia , Biossíntese de Proteínas , Reação em Cadeia da Polimerase em Tempo Real , Células-Tronco/imunologia
13.
Biomaterials ; 33(16): 4204-19, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22417621

RESUMO

Development of efficient and safe nucleic acid carriers (vectors) is one of the essential requirements for the success of gene therapy. Here, we have evaluated the gene transfer capability of chitosan-PEI (CP) conjugates prepared by conjugating low molecular weight branched polyethylenimine (LMWP) with depolymerized chitosans (7 and 10 kDa) via their terminal aldehyde/keto groups. The CP conjugates interacted efficiently with nucleic acids and also showed higher cellular uptake. These conjugates on complexation with DNA yielded nanoparticles in the size range of 100-130 nm (in case of C7P) and 115-160 nm (in case of C10P), which exhibited significantly higher transfection efficiency (~2-42 folds) in vitro compared to chitosans (high and low mol. wt.) and the commercially available transfection reagents retaining cell viability almost comparable to the native chitosan. Of the two CP conjugates, chitosan 7 kDa-LMWP (C7P) displayed higher gene transfer ability in the presence and absence of serum. Luciferase reporter gene analysis in male Balb/c mice receiving intravenous administration of C7P3/DNA polyplex showed the maximum expression in their spleen. Further, tuftsin, a known macrophage targeting molecule, was tethered to C7P3 and the resulting complex, i.e., C7P3-T/DNA, exhibited significantly higher gene expression in cultured mouse peritoneal macrophages as compared to unmodified C7P3/DNA complex without any cytotoxicity demonstrating the suitability of the conjugate for targeted applications. Conclusively, the study demonstrates the potential of the projected conjugates for gene delivery for wider biomedical applications.


Assuntos
Quitosana/química , Macrófagos/efeitos dos fármacos , Ácidos Nucleicos/farmacologia , Polietilenoimina , Polímeros/química , Tuftsina/química , Animais , Linhagem Celular , Citometria de Fluxo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Ácidos Nucleicos/administração & dosagem , Tamanho da Partícula , RNA Interferente Pequeno
14.
Free Radic Res ; 45(9): 1061-73, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21726175

RESUMO

Kolaviron (KV), a natural biflavonoid obtained from the seeds of Garcinia kola, has been documented for its wide pharmacological window, including anti-apoptotic activities. However, the underlying mechanisms are poorly understood at the cellular level. This study investigates the anti-apoptotic activity of KV in PC12 cells, a rat pheochromocytoma, exposed to endocrine disruptor-atrazine (ATZ). KV (60 µM) treatment for 24 h shows significant anti-apoptotic responses in PC12 cells exposed to ATZ (232 µM) for 24 h. KV treatment recovers the ATZ-induced levels of malondialdehyde, reactive oxygen species (ROS), caspase-3 activity and depleted levels of glutathione and catalase activity. However, KV was found to be ineffective to restore the ATZ-induced expression (mRNA) and activity of glutathione-peroxidase (GSH-Px) and glutathione reductase (GR). KV treatment also demonstrates significant restoration in ATZ-induced alterations in the expression of apoptosis markers viz., p53, Bax, Bcl2, caspase-3, caspase-9, cyclooxygenase-2 (COX-2), c-Jun and c-fos. Flow cytometric analysis confirms the involvement of ROS in the mediation of ATZ-induced apoptosis in PC12 cells. Together, these data suggest that KV has the therapeutic potential against chemical-induced apoptotic cell death in the neuronal system.


Assuntos
Apoptose/efeitos dos fármacos , Atrazina/antagonistas & inibidores , Citoproteção , Flavonoides/farmacologia , Neurônios/efeitos dos fármacos , Animais , Atrazina/química , Atrazina/toxicidade , Caspase 3/análise , Catalase/análise , Linhagem Celular Tumoral , Disruptores Endócrinos/toxicidade , Flavonoides/química , Glutationa/análise , Glutationa Peroxidase/metabolismo , Glutationa Redutase/metabolismo , Malondialdeído/análise , Neurônios/fisiologia , Células PC12 , Ratos , Espécies Reativas de Oxigênio/análise
15.
Eur J Pharmacol ; 666(1-3): 5-11, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21620820

RESUMO

In this study, we determined the protective potential of trans resveratrol against oxygen-glucose deprivation (OGD) induced reactive oxygen species mediated apoptotic damages in PC12 cells. In vitro model of ischemic cerebral stroke was created by keeping cells in an OGD condition for 6h followed by 24h reoxygenation. Cells received biologically safe doses (5, 10, and 25 µM) of trans resveratrol in the following schedules for 24h prior to OGD; during 6h of OGD; for 24h post OGD and whole treatment group which starts from 24h before OGD and lasted to 24h post OGD. Anti-ischemic potential of trans resveratrol was assessed by measuring the regulation of lipid peroxidation, reactive oxygen species production, glutathione content, and expression (mRNA and protein) of apoptotic markers such as Bax, Bcl(2) and Caspase-3. Hypoxia inducible factor-1α (HIF-1α) was also assessed to correlate the changes with ischemic injuries. Significant (P<0.05) restoration in lipid peroxidation, reactive oxygen species, and glutathione content were observed following the treatment of trans resveratrol in cells receiving OGD and re-oxygenation. Changes induced by trans resveratrol could be correlated well with alterations in the expression of Bax, Bcl(2), Caspase-3 and HIF-1α. These results indicate that trans resveratrol administration attenuates free radical formation and mitochondria mediated apoptosis perhaps by regulating the expressions of Bax, Bcl(2,) and Caspase-3 in PC12 cells receiving OGD and re-oxygenation insult.


Assuntos
Apoptose/efeitos dos fármacos , Isquemia Encefálica/patologia , Fármacos Neuroprotetores/farmacologia , Estilbenos/farmacologia , Animais , Apoptose/genética , Isquemia Encefálica/genética , Isquemia Encefálica/metabolismo , Isquemia Encefálica/prevenção & controle , Caspase 3/biossíntese , Caspase 3/genética , Glucose/metabolismo , Glutationa/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Peroxidação de Lipídeos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Oxigênio/metabolismo , Células PC12 , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , Ratos , Espécies Reativas de Oxigênio/metabolismo , Resveratrol , Transcrição Gênica/efeitos dos fármacos , Proteína X Associada a bcl-2/biossíntese , Proteína X Associada a bcl-2/genética
16.
Nanotoxicology ; 5(2): 195-207, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20804439

RESUMO

Multi-walled carbon-nanotubes (MWCNTs)-induced apoptotic changes were studied in human lung epithelium cell line-A549. Non-cytotoxic doses of MWCNTs were identified using tetrazolium bromide salt (MTT) and lactate dehydrogenase (LDH) release assays. Cells were exposed to MWCNTs (0.5-100 µg/ml) for 6-72 h. Internalization and characterization of CNTs was performed by electron microscopy. Apoptotic changes were estimated by nuclear condensation, DNA laddering, and confirmed by expression of associated markers: p(53), p(21WAF1/CIP1), Bax, Bcl(2) and activated caspase-3. MWCNTs induced the production of reactive oxygen species and malondialdehyde along with significant decrease in the activity of catalase and glutathione. MWCNTs-induced ROS generation was found not to be associated with the mitochondrial activity. In general, the changes were significant at 10 and 50 µg/ml only. Results indicate the involvement of oxidative stress and apoptosis in A549 cells exposed to MWCNTs. Our studies provide insights of the mechanisms involved in MWCNTs-induced apoptosis at cellular level.


Assuntos
Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Nanotubos de Carbono/efeitos adversos , Estresse Oxidativo/efeitos dos fármacos , Animais , Biomarcadores/metabolismo , Humanos , Neoplasias Pulmonares , Mitocôndrias/metabolismo , Nanotubos de Carbono/ultraestrutura , Espécies Reativas de Oxigênio/metabolismo , Rotenona/metabolismo , Desacopladores/metabolismo
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