RESUMEN
The edible endosperm of Areca catechu is recognized as a potent carcinogenic agent either consumed alone or in combination with tobacco. Habitual chewing of areca nut leads to orally potential malignant disorders which are highly effective in malignant transformation and thereby lead to oral carcinogenesis. Human buccal epithelial KB carcinoma cells were used as an experimental cell system to inspect the mechanistic act of aqueous extract of areca nut on biochemical status and their implications on transcriptional activation of cancer signaling cascade that could possibly trigger numerous oncogenic players and finally decides the cells fate. Extract treated cells showed reduced viability with altered balance between oxidants and antioxidants which lead to redox status and which is known to distort various biological processes within the cell system. Results of RT-PCR demonstrated decreased expression of BCl2, cell cycle regulators along with Activator Protein -1 (AP-1) components. While Bax, p16 and p21 mRNAs showed increased expression in extract treated KB cells. Likewise, the translational levels of proliferation cell nuclear antigen (PCNA), tumor suppressor p53, retinoblastoma (Rb) and cyclin dependent kinase 4 (CDK4) were decreased along with AP-1 subunits (c-Jun/c-Fos) with increased protein levels of p21 in extract treated KB cells. Further, the downstream activation and regulation of AP-1 transcription factors could be through stress activated c-Jun - N terminal Kinase (JNK) Mitogen Activated Protein Kinases (MAPKs) which downregulated both Jun and Fos mRNA transcripts in areca nut extract exposed KB cells. Thus, outcome of the study provides insights into mechanistic path of pathogenesis of areca related disorders. Further, it could aid in designing new therapeutic modalities that specific targets these oncogenic players and help in disease management.
RESUMEN
Activator protein-1 (AP-1) transcription factor is a key component of many signal transduction pathways involved in the regulation of cellular processes and controls rapid responses of mammalian cells when exposed to the variety of stimulus. The phorbol 12-myristate 13-acetate and Forskolin (Fo) are well-known kinase activators/stimulators of Protein Kinase C (PKC) and Protein Kinase A (PKA) respectively. Importantly, these kinases are found to be present in transitional points of many cell signaling pathways, especially those involved in proliferation. The stimulating effect of PKC and PKA on the expression of AP-1 factors in MCF-7 breast cell proliferation is not well characterized. Hence, the role of PKC by PMA treatment and the role of PKA by using Fo in MCF-7 cells is investigated. Where, cells treated with PMA showed increased cell proliferation, while Fo had no effect, but inhibited the PMA induced proliferation. The RT-PCR results showed the PMA induced c-Jun, c-Fos and Fra-1 expressions compared to control and Fo. However, Fo in combination with PMA, inhibit the PMA induced above mRNA expressions where Fo alone has no effect. Western blot studies validated the c-Jun expressions in PMA treated MCF-7 cells. Further, PMA increases the mRNA expression of Cyclin-E1, Cyclin-D1, and CDK-4, whereas Fo decreases their expressions. Thus, mitogenic effect of PMA and inhibitory action of Fo on MCF-7 cells is probably enhanced via activation of AP-1 factors and concomitant action of cell cycle regulators in the downstream singling cascade.
RESUMEN
Areca nut chewing habits are associated with several oral manifestations like leukoplakia, submucous fibrosis and oral squamous cell carcinoma. Although numerous evidence on areca toxicity is known but the mechanistic pathway of disease causation is to be studied. Aqueous areca nut extract treated A549 cells showed reduced cell viability by 48 h with IC50 value of 0.50%. The toxic nature of areca nut induced the production of reactive oxygen species with decreased anti-oxidant glutathione S transferase levels lead to altered redox homeostasis. PCR studies showed decreased mRNA levels of Jun and Fos AP-1 subunits on extract treatment by 48 h. The protein levels of PCNA, CDK4, RB, p53, c-Jun and c-Fos were found to be downregulated with upregulated CDK inhibitor p21 on extract treatment as compared to control. Results of FACS analysis further confirm G1/S phase cell cycle arrest on areca nut extract exposure. The regulation of downstream AP-1 subunits by MAPKs was studied by using specific inhibitors of ERK, JNK and p38 along with areca nut extract. Results showed the redox activation of MAP kinases down regulated the mRNA levels of AP-1 subunits in aqueous areca nut extract treated cells. Hence the present study aids in elucidating the role of MAP kinases in regulating the AP-1 subunits and their implications on target genes that are involved regulation of various cellular processes. Further, it would help in understanding the mechanistic aspects of the diseased state which may facilitate in designing of new therapeutic modalities that could help in cancer management.
RESUMEN
Choriocarcinoma, a trophoblastic neoplasia, occurs in women as an incidence of abnormal pregnancy. BeWo choriocarcinoma cells derived from the abnormal placentation are a suitable model system to study the factors associated with differentiation, invasion and other cellular events as an alternative to clinical samples. Many protein kinases orchestrate the complex events of cell cycle and in case of malignancy such regulators are found to be mutated. In the present study, BeWo cells treated with forskolin (Fo) and phorbol 12-myristate 13-acetate (PMA) were used to study the role of PKA (protein kinase A) and PKC (protein kinase C), respectively, on the expression pattern of differentiation-related genes, membrane markers, PKC isoforms and cell cycle regulators. The effect of Fo and PMA on the cell proliferation was assessed. Progressive induction of alkaline phosphatase level and formation of multinucleated differentiated cells were observed in the cells treated with Fo. Exposure of cells to Fo and PMA induced the mRNA transcripts of α-hCG, ß-hCG and endoglin and down-regulates E-cadherin at mRNA and protein levels. Synergistic levels of both up- and down-regulated genes/proteins were observed when cells were treated with the combination of Fo and PMA. The mRNA levels of cyclin D1, cyclin E1, p21, Rb, p53, caspase-3 and caspase-8 decreased gradually during differentiation. Fo significantly inhibited the protein levels of PCNA, Rb, PKC-α and PMA stimulated mRNA expression of PKC-ε and PKC-δ. Further, failure in the activation of essential components of the cell cycle machinery caused G2/M phase arrest in differentiating BeWo cells.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Diferenciación Celular , Coriocarcinoma/patología , Proteínas Quinasas/metabolismo , Proteínas de Ciclo Celular/genética , Proliferación Celular , Coriocarcinoma/enzimología , Humanos , Proteínas Quinasas/genética , Células Tumorales CultivadasRESUMEN
The production of inflammatory mediators by epithelial cells in inflammatory lung diseases may represent an important target for the anti-inflammatory effects of glucocorticoids. Activator protein-1 is a major activator of inflammatory genes and has been proposed as a target for inhibition by glucocorticoids. We have used human pulmonary type-II A549 cells to examine the effect of dexamethasone on the phorbol ester (PMA)/Lipopolysaccharide (LPS) induced pro-inflammatory cytokines and AP-1 factors. A549 cells were treated with and without PMA or LPS or dexamethasone and the cell viability and nitric oxide production was measured by MTT assay and Griess reagent respectively. Expression of pro-inflammatory cytokines and AP-1 factors mRNA were measured using semi quantitative RT-PCR. The PMA/LPS treated cells show significant 2-3 fold increase in the mRNA levels of pro-inflammatory cytokines (IL-1ß, IL-2, IL-6, IL-8 and TNF-α), cyclooxygenase-2 (COX-2) and specific AP-1 factors (c-Jun, c-Fos and Jun-D). Whereas, pretreatment of cells with dexamethasone significantly inhibited the LPS induced nitric oxide production and PMA/LPS induced mRNAs expression of above pro-inflammatory cytokines, COX-2 and AP-1 factors. Cells treated with dexamethasone alone at both the concentrations inhibit the mRNAs expression of IL-1ß, IL-6 and TNF-α compared to control. Our study reveals that dexamethasone decreased the mRNAs expression of c-Jun and c-Fos available for AP-1 formation suggested that AP-1 is the probable key transcription factor involved in the anti-inflammatory activity of dexamethasone. This may be an important molecular mechanism of steroid action in asthma and other chronic inflammatory lung diseases which may be useful for treatment of lung inflammatory diseases.
Asunto(s)
Antiinflamatorios/farmacología , Dexametasona/farmacología , Regulación hacia Abajo , Pulmón/efectos de los fármacos , Factor de Transcripción AP-1/genética , Células A549 , Supervivencia Celular/efectos de los fármacos , Citocinas/genética , Células Epiteliales/química , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Lipopolisacáridos/efectos adversos , Pulmón/química , Pulmón/citología , Pulmón/inmunología , Óxido Nítrico/metabolismo , Ésteres del Forbol/efectos adversosRESUMEN
Androgen receptor (AR) signaling axis plays a vital role in the development of prostate and critical in the progression of prostate cancer. Androgen withdrawal initially regresses tumors but eventually develops into aggressive castration-resistant prostate cancer (CRPC). Activator Protein-1 (AP-1) transcription factors are most likely to be associated with malignant transformation in prostate cancer. Hence, to determine the implication of AR and AP-1 in promoting the transition of prostate cancer to the androgen-independent state, we used AR-positive LNCaP and AR-negative PC-3 cells as an in vitro model system. The effect of dihydrotestosterone or anti-androgen bicalutamide on the cell proliferation and viability was assessed by MTT assay. Expression studies on AR, marker genes-PSA, TMPRSS2, and different AP-1 factors were analyzed by semi-quantitative RT-PCR and expressions of AR and Fra-1 proteins were analyzed by Western blotting. Dihydrotestosterone induced the cell proliferation in LNCaP with no effect on PC-3 cells. Bicalutamide decreased the viability of both LNCaP and PC-3 cells. Dihydrotestosterone induced the expression of AR, PSA, c-Jun, and Fra-1 in LNCaP cells, and it was c-Jun and c-Fos in case of PC-3 cells, while bicalutamide decreased their expression. In addition, constitutive activation and non-regulation of Fra-1 by bicalutamide in PC-3 cells suggested that Fra-1, probably a key component, involved in transition of aggressive androgen-independent PC-3 cells with poor prognosis.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Factor de Transcripción AP-1/metabolismo , Línea Celular Tumoral , Humanos , Masculino , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/patología , Proteínas Proto-Oncogénicas c-fos/genética , Factor de Transcripción AP-1/genéticaRESUMEN
Cadmium (Cd) is one of the well-known highly toxic environmental and industrial pollutants. Cd first accumulates in the nucleus and later interacts with zinc finger proteins of antiapoptotic genes and inhibit the binding of transcriptional factors and transcription. However, the role of Cd in oxidative stress and apoptosis is less understood. Hence, the present study was undertaken to unveil the mechanism of action. A549 cells were treated with or without Cd and cell viability was measured by MTT assay. Treatment of cells with Cd shows reduced viability in a dose-dependent manner with IC50 of 45 µM concentration. Cd significantly induces the reactive oxygen species (ROS), lipid peroxidation followed by membrane damage with the leakage of lactate dehydrogenase (LDH). Cells with continuous exposure of Cd deplete the antioxidant super oxide dismutase (SOD) and glutathione peroxidase (GSH-Px) enzymes. Further, analysis of the expression of genes involved in apoptosis show that both the extrinsic and intrinsic apoptotic pathways were involved. Death receptor marker tumor necrosis factor-α (TNF-α), executor caspase-8 and pro-apoptotic gene (Bax) were induced, while antiapoptotic gene (Bcl-2) was decreased in Cd-treated cells. Fluorescence-activated cell sorting (FACS) analysis further confirms the induction of apoptosis in Cd-treated A549 cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Cadmio/toxicidad , Contaminantes Ambientales/toxicidad , Células Epiteliales/efectos de los fármacos , Pulmón/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Células A549 , Antioxidantes/metabolismo , Técnicas de Cultivo de Célula , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Células Epiteliales/metabolismo , Células Epiteliales/patología , Citometría de Flujo , Humanos , Peroxidación de Lípido/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Apigenin is one of the plant flavonoids present in fruits and vegetables, acting as an important nutraceutical component. It is recognized as a potential antioxidant, antimicrobial, and anti-inflammatory molecule. In the present study, the mechanism of anti-inflammatory action of apigenin on lipopolysaccharide (LPS)-induced pro-inflammatory cytokines and activator protein-1 (AP-1) factors in human lung A549 cells was investigated. The anti-inflammatory activity of apigenin on LPS-induced inflammation was determined by analyzing the expression of pro-inflammatory cytokines, nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and different AP-1 factors. Apigenin significantly inhibited the LPS-induced expression of iNOS, COX-2, expression of pro-inflammatory cytokines (IL-1ß, IL-2, IL-6, IL-8, and TNF-α), and AP-1 proteins (c-Jun, c-Fos, and JunB) including nitric oxide production. Study confirms the anti-inflammatory effect of apigenin by inhibiting the expression of inflammatory mediators and AP-1 factors involved in the inflammation and its importance in the treatment of lung inflammatory diseases.
Asunto(s)
Antiinflamatorios/farmacología , Antioxidantes/farmacología , Apigenina/farmacología , Células Epiteliales/metabolismo , Mucosa Respiratoria/metabolismo , Factor de Transcripción AP-1/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Ciclooxigenasa 2/biosíntesis , Citocinas/biosíntesis , Citocinas/genética , Humanos , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Lipopolisacáridos/farmacología , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa de Tipo II/biosíntesis , ARN Mensajero/genética , Mucosa Respiratoria/citologíaRESUMEN
Acute and chronic alveolar or bronchial inflammation is thought to be central to the pathogenesis of many respiratory disorders. Cytokines and granulocyte macrophage colony-stimulating factors (GM-CSF) play an important role in chronic inflammation. Activator protein-1 (AP-1) the superfamily of transcription factors is involved in proliferation, differentiation, apoptosis, and transformation including inflammation. Understanding the function and regulation of proinflammatory factors involved in inflammation may provide the novel therapeutic strategies in the treatment of inflammatory diseases. Our aim of the present study is to investigate the pro-inflammatory cytokines and pattern of AP-1 factors expressed during activation of lung adenocarcinoma A549 cells by Phorbol-12-myristate-13-acetate (PMA) and to understand the anti-inflammatory effect of apigenin. A549 cells were treated with and without PMA or apigenin, and the cell viability was assessed by MTT assay. Expressions of inflammatory mediators and different AP-1 factors were analyzed by semi-quantitative RT-PCR. IL-6 protein secreted was analyzed by ELISA, and expressions of IL-1ß, c-Jun, and c-Fos proteins were analyzed by Western blotting. Activation of A549 cells by PMA, induced the expression of pro-inflammatory cytokine (IL-1ß, IL-2, IL-6, IL-8, and TNF-α) mRNAs and secretion of IL-6 and the expression of specific AP-1 factors (c-Jun, c-Fos, and Fra-1). Treatment of cells with apigenin, significantly inhibited PMA-stimulated mRNA expression of above pro-inflammatory cytokines, AP-1 factors, cyclooxygenase-2, and secretion of IL-6 protein. Results suggested that the AP-1 factors may be involved in inflammation and apigenin has anti-inflammatory effect, which may be useful for therapeutic management of lung inflammatory diseases.