RESUMO
Senescence-associated secretory phenotype (SASP) is often a concomitant result of cell senescence, embodied by the enhanced function of secretion. The SASP factors secreted by senescent cells include cytokines, proteases and chemokines, etc, which can exert great influence on local as well as systemic environment and participate in the process of cell senescence, immunoregulation, angiogenesis, cell proliferation and tumor invasion, etc. Relative to the abundance of SASP models in human cells, the in vitro SASP model derived from mouse cells is scarce at present. Therefore, the study aimed to establish a mouse SASP model to facilitate the research in the field. With this objective, we treated the INK4a-deficient mouse NIH-3T3 cells and the wildtype mouse embryonic fibroblasts (MEF) respectively with mitomycin C (MMC), an anticarcinoma drug which could induce DNA damage. The occurring of cell senescence was evaluated by cell morphology, ß-gal staining, integration ratio of EdU and Western blot. Quantitative RT-PCR and ELISA were used to detect the expression and secretion of SASP factors, respectively. The results showed that, 8 days after the treatment of NIH-3T3 cells with MMC (1 µg/mL) for 12 h or 24 h, the cells became enlarged and the ratios of ß-gal-positive (blue-stained) cells significantly increased, up to 77.4% and 90.4%, respectively. Meanwhile, the expression of P21 protein increased and the integration ratios of EdU significantly decreased (P < 0.01). Quantitative RT-PCR detection showed that the mRNA levels of several SASP genes, including IL-6, TNF-α, IL-1α and IL-1ß increased evidently. ELISA detection further observed an enhanced secretion of IL-6 (P < 0.01). On the contrary, although wildtype MEF could also be induced into senescence by MMC treatment for 12 h or 24 h, embodied by the enlarged cell volume, increased ratios of ß-gal-positive cells (up to 71.7% and 80.2%, respectively) and enhanced expression of P21 protein, the secretion of IL-6 displayed no significant change. Our study indicated that, although MMC could induce senescence in both mouse NIH-3T3 cells and wildtype MEF, only senescent NIH-3T3 cells displayed the canonical SASP phenomena. Current study suggested that senescent NIH-3T3 cells might be an appropriate in vitro SASP model of mouse cells.
Assuntos
Senescência Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Mitomicina/farmacologia , Animais , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Citocinas/genética , Citocinas/metabolismo , Dano ao DNA , Interleucina-6/metabolismo , Camundongos , Células NIH 3T3 , FenótipoRESUMO
In this paper, a novel electrochemiluminescence resonance energy transfer (ECL-RET) system from O2/S2O8(2-) to a kind of amino-terminated perylene derivative (PTC-NH2) was demonstrated for the first time, which was then applied to construct a ratiometric aptasensor for lead ion (Pb(2+)) detection. First, gold-nanoparticles-functionalized fullerene nanocomposites (AuNPs@nano-C60) were coated on a glassy carbon electrode (GCE), and then thiol-modified assistant probes (APs) were attached on AuNPs@nano-C60/GCE. Then the resultant electrode was hybridized with capture probes (the aptamer of the Pb(2+), abbreviated as CPs) to generate DNA duplexes, which could induce PTC-NH2 to be intercalated into the dsDNA grooves by the electrostatic adsorption. Herein, ECL dual peaks at -0.7 V (vs Ag/AgCl) and -2.0 V (vs Ag/AgCl) were obtained when the prepared aptasensor was detected in air-saturated S2O8(2-) solution, which could be attributed to the emission of excited dimmers (π-excimers) ((1)(NH2-PTC)2*) and (1)(O2)2*, respectively. In the presence of Pb(2+), the dsDNA was unwound, and Pb(2+) G-quadruplex structure was generated because of the highly specific affinity between Pb(2+) and CPs, which made the PTC-NH2 release from the electrode surface. As a result, the ECL signal at -0.7 V was decreased, and the ECL signal around -2.0 V was increased. By measuring the ratio of ECL intensities at two excitation potentials, the developed aptasensor exhibited the linear response range from 1.0 × 10(-12) M to 1.0 × 10(-7) M with a detection limit of 3.5 × 10(-13) M (S/N = 3) for Pb(2+), which could offer an alternative analytical method with excellent properties of high selectivity, accuracy, and sensitivity.
Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas , Chumbo/análise , Ouro/química , Íons/análise , Medições Luminescentes , Nanopartículas Metálicas/química , Solo/químicaRESUMO
OBJECTIVE: To explore the anti-tumor effects of resveratrol (Res) upon human skin squamous cell carcinoma A431 xenograft in nude mice and elucidate the regulatory mechanisms of survivin and caspase-3. METHODS: The model of human skin squamous cell carcinoma (A431) xenograft in nude mice was established. And the animals were randomly divided into saline-negative control, cyclophosphamide (CTX) positive control, Res high-, medium- and low-dosage and blank control groups (n = 10 each). After drug intervention, tumor-bearing mice were sacrificed. The tumor growth curve was plotted and the Res inhibition rate calculated by terminal tumor weight. The morphological changes of tumor cell among groups were observed by hematoxylin and eosin staining; cell apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling TUNEL; the impact of Res upon the protein expressions of survivin and caspase-3 in tumor issues was observed by Western blot. Analysis of variance and Pearson's correlation were employed for statistical analyses. RESULTS: (1) By the end of treatment, the tumor volume of CTX, Res high-, medium-, low-dosage, saline-negative control and blank control groups were (1154 ± 255), (1002 ± 115), (1207 ± 176), (1342 ± 211), (1642 ± 226), (1564 ± 156) mm(3) respectively, and tumor weight of CTX, Res high-, medium-, low-dosage, saline-negative control and blank control groups (1.84 ± 0.30), (1.72 ± 0.39), (1.96 ± 0.40), (2.67 ± 0.73), (3.16 ± 0.52), (3.33 ± 0.59) g respectively. Through analysis of variance, the tumor volume and weight of Res high-, medium-, low-dosage groups were smaller than those of saline-negative control and blank control groups (all P < 0.05). The inhibition rate of Res high-, medium- and low-dosage groups were 45.57%, 37.97% and 15.51% respectively. (2) The apoptosis index of the above groups were 36.79% ± 8.86%, 33.15% ± 6.00%, 18.09% ± 3.92%, 10.53% ± 4.20%, 3.87% ± 1.63%, 2.73% ± 1.61%. Through analysis of variance, the apoptosis index of Res groups were higher than those of saline-negative control and blank control groups (all P < 0.05). (3) The protein expression of survivin/ß-actin of each group were 0.48 ± 0.20, 0.19 ± 0.11, 0.22 ± 0.12, 0.28 ± 0.24, 0.98 ± 0.41, 0.85 ± 0.34. The protein expression of caspase-3/ß-actin of each group were 0.42 ± 0.09, 0.31 ± 0.10, 0.31 ± 0.07, 0.22 ± 0.08, 0.14 ± 0.04, 0.13 ± 0.05 respectively. Through analysis of variance, the protein expression of survivin of Res groups was lower than those of the saline-negative control and blank control groups (all P < 0.05). And the protein expression of caspase-3 of Res groups were higher than those of the saline-negative control and blank control group (all P < 0.05). Through Pearson's analysis, the protein expression of survivin and caspase-3 had no correlation (r = -0.279, P > 0.05). CONCLUSIONS: Res inhibits the growth of human skin squamous cell carcinoma A431 xenograft in nude mice. And its mechanism may be associated with the apoptosis of tumor cell through the depression of survivin and the activation of caspase-3.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Neoplasias Cutâneas/metabolismo , Estilbenos/farmacologia , Animais , Apoptose , Caspase 3/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Camundongos , Camundongos Nus , Resveratrol , Survivina , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
AIM: The progression of atherosclerosis can lead to the occurrence of multiple cardiovascular diseases (coronary heart disease, etc.). E prostanoid receptor-3 (EP3) is known to participate in the progression of atherosclerosis. This study aimed to investigate the mechanism by which EP3 modulates the development of atherosclerosis. METHODS AND RESULTS: ApoE-/- mice were used to construct in vivo model of atherosclerosis. Human aortic smooth muscle cells (HASMCs) were stimulated with oxidized low-density lipoprotein (ox-LDL) to construct in vitro model of atherosclerosis. mRNA expressions were assessed by qRT-PCR, and western blot was applied to assess the protein levels. CCK-8 assay was applied to assess the cell viability. The inflammatory cytokines levels were assessed by enzyme-linked immunosorbent assay, and flow cytometry was applied to assess cell apoptosis. In vivo experiment was constructed to investigate the impact of EP3 in atherosclerosis development. L-798106 (EP3 inhibitor) significantly inhibited the levels of pro-inflammatory cytokines in atherosclerosis in vivo. EP3 inhibitor (L-798106) significantly reversed ox-LDL-caused HASMCs injury via inhibiting the apoptosis and inflammatory responses (P < 0.05). The levels of interleukin-17 (IL-17) and intercellular adhesion molecule-1 (ICAM-1) in HASMCs were elevated by ox-LDL, whereas L-798106 or knockdown of cyclic AMP (cAMP) response element-binding protein (CREB) notably restored this phenomenon (P < 0.05). EP3 overexpression further aggravated ox-LDL-induced inflammation in HASMCs, and EP3 up-regulated the levels of IL-17 and ICAM-1 in ox-LDL-treated HASMCs (P < 0.05). EP3 up-regulation promoted the inflammatory responses in ox-LDL-treated HASMCs through mediation of cAMP/protein kinase A (PKA)/CREB/IL-17/ICAM-1 axis (P < 0.05). CONCLUSIONS: EP3 inhibitor alleviates ox-LDL-induced HASMC inflammation via mediation of cAMP/PKA/CREB/IL-17/ICAM-1 axis. Our study might shed new lights on discovering novel strategies against atherosclerosis.