Effect of CASC2a on proliferation and migration of papillary thyroid carcinoma cells via NF-κB signaling pathway / 医学研究生学报

Objective There are few reports on the relationship between lncRNA cancer susceptibility candidate gene 2 (CASC2) and NF-κB signaling pathway in thyroid papillary carcinoma cells at home and abroad. This study aimed to investigate the effect of lncRNA CASC2a on the proliferation and migration of TPC-1 via NF-κB signaling pathway. Methods TPC-1 was selected and constructed into lncRNA CASC2a overexpression plasmids which were divided into plasmid group [transfection with overexpressed plasmid pcDNA3.1(+)-CASC2a], empty group [transfection with equal amount of pcDNA3.1 (+) empty plasmid], blank group (without any processing). The effect of overexpressed lncRNA CASC2a on the expression of CASC2a in each group of TPC-1 cells was examined. The cells of transfected plasmid group were randomly divided into transfection group [transfection with only overexpressed plasmid pcDNA3.1(+)-CASC2a], transfection + PMA group [transfection with overexpressed plasmid pcDNA3.1(+)- CASC2a + propylene glycol methyl ether acetate (PMA) stimulation], control group (without any processing), PMA group (only plus PMA stimulation). The effects of lncRNA CASC2a on cell proliferation and migration were verified by CCK-8 and cell scratch assays at 24, 48, 72 and 96 h. Western blot was used to detect the expression of p105/p50 and p65 in NF-κB signaling pathway. The expression of NF-κB signaling pathway-related antibody proteins p105/p50 and p65 was observed by NF-κB signaling pathway agonist PMA(propylene glycol methyl ether acetate). Results After transfection with overexpressed plasmid pcDNA3.1(+)-CASC2a, the expression of CASC2a in plasmid group was significantly higher than that in empty group (P<0.05). The cell proliferation ability (0.191±0.005) was significantly lower in transfection+PMA group than that in transfection group (0.217±0.013), PMA group (0.247±0.009), and control group (0.260±0.004), and the difference was statistically significant ( P<0.01), while the cell proliferation ability in transfection group was significantly lower than those in PMA group and control group (P<0.01). The cell migration ability of transfected + PMA group [(0.208±0.109)%] was lower than that of transfection group, PMA group, and control group [(1.775±0.061)%, (1.622±0.519)%, (2.927±0.136)%], while the cell migration ability of transfection group and PMA group was lower than that in control group (P<0.05). The relative expression of p105/p50 and p65 protein in plasmid group was significantly lower than that in blank group (P<0.05). The expression of p105/p50 and p65 protein in transfection+PMA group [(0.674±0.007), (0.713±0.014)] was significantly higher than that in transfection group [(0.581±0.003), (0.570±0.012)] (P<0.05). Conclusion lncRNA CASC2a may inhibit the proliferation and migration of thyroid papillary carcinoma cells through NF-κB signaling pathway.

Phenotypic characterization of PMA-induced THP-1 macrophages analyzed by flow cytometry / 中国比较医学杂志

Objective To identify the characteristics of the subtype of PMA-induced THP-1 macrophages by flow cytometry analysis. Methods THP-1 monocytic cells differentiate into macrophages promoted by PMA, then induced into M1 and M2 by adding different cytokines, such as LPS,IL-6 and IFN-γ for THP-1-M1, IL-4,IL-13 and IL-6 for THP-1-M2. Morphology of cells were observed under a microscope and the expression of CD14, CD68, CD16, CD80, CD86, CD163, CD206, CD209, CD83, CD1a, CD11c, HLA-DR were detected by flow cytometry. Results The macrophages stimulated by PMA became adherent;THP-1-M1 and THP-1-M2 lost their spherical morphology, appeared more irregular with many obvious projections. The expression of CD83, CD1a, CD11c, HLA-DR which had the function of antigen presenting on the surface of THP-1-Mφwere very low, and most of them were negative, but those of THP-1-M1 and THP-1-M2 were very high. Conclusions The macrophages differentiated from THP-1 stimulated by PMA are weak in antigen presenting function.

Phenotypic characterization of PMA-induced THP-1 macrophages analyzed by flow cytometry / 中国比较医学杂志

Objective To identify the characteristics of the subtype of PMA-induced THP-1 macrophages by flow cytometry analysis. Methods THP-1 monocytic cells differentiate into macrophages promoted by PMA, then induced into M1 and M2 by adding different cytokines, such as LPS,IL-6 and IFN-γ for THP-1-M1, IL-4,IL-13 and IL-6 for THP-1-M2. Morphology of cells were observed under a microscope and the expression of CD14, CD68, CD16, CD80, CD86, CD163, CD206, CD209, CD83, CD1a, CD11c, HLA-DR were detected by flow cytometry. Results The macrophages stimulated by PMA became adherent;THP-1-M1 and THP-1-M2 lost their spherical morphology, appeared more irregular with many obvious projections. The expression of CD83, CD1a, CD11c, HLA-DR which had the function of antigen presenting on the surface of THP-1-Mφwere very low, and most of them were negative, but those of THP-1-M1 and THP-1-M2 were very high. Conclusions The macrophages differentiated from THP-1 stimulated by PMA are weak in antigen presenting function.

Effects of PKC activation on expression of Nrf2 in rabbit RPE cells / 眼科新进展

Objective To investigate whether activation of protein kmase C (PKC) can induce the activation and nuclear translocation of nuclear factor enthroid 2-related factor 2 (Nrf2) in retinal pigment epithelial (RPE) cells in vitro,and explore whether PKC activation may affect the expression of Nrf2 in RPE cells.Methods PKC-specific activator phorbol ester PMA was used to culture rabbit RPE cells and RPE cells pretreated with Nrf2 inhibitor for 24 hours.Immunofluorescence and Western blot were used to detect Nrf2 in the nucleus of the expression of the situation,the data were obtained for statistical analysis.Results The expression of Nrf2 protein in the nucleus of PRE cells was detected by immunofluorescence.Compared with the control group,the expression of Nrf2 protein in the nucleus of RPE cells increased in the experimental group,and the increase of PMA + Nrf2 inhibitor group was lower than that of PMA group.The difference between the two groups was statistically significant (P ＜0.05).Western blot analysis showed that the Nrf2 protein in the nucleus of PRE was quantitatively analyzed by image analysis.The gray value of the control group was significantly different (0.286 ± 0.013 in the control group,1.304 ± 0.033 in the PMA group and 0.671 ± 0.087 in the PMA + Nrf2 inhibitor group,P ＜ 0.05).Conclusion PKC can activate nuclear translocation of Nrf2 in rabbit RPE cytoplasm,and Nrf2 inhibitor can attenuate the effect of PKC.

Sustained small and intermediate size proteins expression in phorbol 12-myristate 13-acetate/ionomycine prolonged stimulated human fibroblasts

Objective:To compare the protein profile of culture supematants in stimulated and unstimulated human fibroblasts to find some proteins indicating the presence of fibroblasts and their activation status.Methods:Dcrmal fibroblasts were stimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycine for 72 h.MTT assay was done to determine cell viability and A/E fluorescent staining was used to evaluate the cell death pattern.Protein analysis was performed by gradient SDS polyacrylamide gel electrophoresis 8％-16％.Results:The supernatant of 24 h cultured both stimulated and unstimulated fibroblasts showed two bands in SDS-PAGE analysis with relative molecular weights of 8.59 and 78,8 kDa.These bands density was decreased during the next 48 h in unstimulated cells while their expression was continued in PMA or PMA/ionomycine stimulated cells and a new 85.3 kDa band was appeared in unstimulated and 72 h PMA stimulated cells.Moreover,we found another seven small size (10-19.5 kDa) proteins in supernatants of 48 h and 72 h unstimulated but not in PMA or PMA/lonomycine stimulated fibroblasts.Most of these proteins expression were down regulated following fibroblast activation.This down-regulation is consistent with our finding that PMA or PMA/ionomycine stimulated cells exhibited a significant level of apoptosis cell death.ConcLusions:Human fibroblasts produce some small to intermediate sized proteins with specific SDS-PAGE profile upon cell activation.Most of these proteins can be excreted in urine and can be immunogen theoretically so this data provided a reliable clue for fibrosis biomarker screening based on designation of an appropriated immunoassay.

Sustained small and intermediate size proteins expression in phorbol 12-myristate 13-acetate/ionomycine prolonged stimulated human fibroblasts

Objective To compare the protein profile of culture supernatants in stimulated and unstimulated human fibroblasts to find some proteins indicating the presence of fibroblasts and their activation status. Methods Dermal fibroblasts were stimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycine for 72 h. MTT assay was done to determine cell viability and A/E fluorescent staining was used to evaluate the cell death pattern. Protein analysis was performed by gradient SDS polyacrylamide gel electrophoresis 8%–16%. Results The supernatant of 24 h cultured both stimulated and unstimulated fibroblasts showed two bands in SDS-PAGE analysis with relative molecular weights of 8.59 and 78.8 kDa. These bands density was decreased during the next 48 h in unstimulated cells while their expression was continued in PMA or PMA/ionomycine stimulated cells and a new 85.3 kDa band was appeared in unstimulated and 72 h PMA stimulated cells. Moreover, we found another seven small size (10–19.5 kDa) proteins in supernatants of 48 h and 72 h unstimulated but not in PMA or PMA/Ionomycine stimulated fibroblasts. Most of these proteins expression were down regulated following fibroblast activation. This down-regulation is consistent with our finding that PMA or PMA/ionomycine stimulated cells exhibited a significant level of apoptosis cell death. Conclusions Human fibroblasts produce some small to intermediate sized proteins with specific SDS-PAGE profile upon cell activation. Most of these proteins can be excreted in urine and can be immunogen theoretically so this data provided a reliable clue for fibrosis biomarker screening based on designation of an appropriated immunoassay.

Acetylshikonin Inhibits Human Pancreatic PANC-1 Cancer Cell Proliferation by Suppressing the NF-kappaB Activity

Acetylshikonin, a natural naphthoquinone derivative compound, has been used for treatment of inflammation and cancer. In the present study, we have investigated whether acetylshikonin could regulate the NF-kappaB signaling pathway, thereby leading to suppression of tumorigenesis. We observed that acetylshikonin significantly reduced proliferation of several cancer cell lines, including human pancreatic PANC-1 cancer cells. In addition, acetylshikonin inhibited phorbol 12-myristate 13-acetate (PMA) or tumor necrosis-alpha (TNF-alpha)-induced NF-kappaB reporter activity. Proteome cytokine array and real-time RT-PCR results illustrated that acetylshikonin inhibition of PMA-induced production of cytokines was mediated at the transcriptional level and it was associated with suppression of NF-kappaB activity and matrix metalloprotenases. Finally, we observed that an exposure of acetylshikonin significantly inhibited the anchorage-independent growth of PANC-1 cells. Together, our results indicate that acetylshikonin could serve as a promising therapeutic agent for future treatment of pancreatic cancer.

Modulatory effects of Azadirachta indica leaf extract on cutaneous and hepatic biochemical status during promotion phase of DMBA/TPA-induced skin tumorigenesis in mice.

The modulation in biochemical status of skin and hepatic tissue at the time point of commencement of promotion stage of skin carcinogenesis in mice and its intervention with aqueous Azadirachta indica leaf extract (AAILE) were investigated. 7,12-Dimethylbenz(a)anthracene (DMBA, 500 nmol/100 ul of acetone) was applied topically for 2 weeks (twice weekly), followed by phorbol-12-myristate-13-acetate (TPA, 1.7 nmol/100 ul) twice weekly for 6 weeks on the depilated skin of mice and AAILE was administered orally at a dose level of 300 mg/kg body wt thrice a week for 10 weeks. DMBA/TPA treatment upregulated the phase I enzymes in skin and hepatic tissue, as revealed by the increased cytochrome P450 (CYP) and cytochrome b5 (cyt b5) levels and aryl hydrocarbon hydroxylase (AHH) activity when compared to the control group and differentially modulated the activities of phase II enzymes like glutathione-s-transferase (GST), DT-diaphorase (DTD) and uridine diphosphate glucuronosyltransferase (UDP-GT). AAILE treatment decreased the DMBA/TPA-induced increase in cutaneous CYP level and enhanced the DTD and UDP-GT activities when compared with DMBA/TPA group. In the hepatic tissue of AAILE + DMBA/TPA group, an increase in UDP-GT activity was observed when compared to DMBA/TPA group. DMBA/TPA treatment did not alter the skin lipid peroxidation (LPO) level when compared to control group, however, in the animals that received AAILE treatment along with DMBA/TPA, a significant increase in LPO was observed when compared to control group. This was associated with a decrease in cutaneous reduced glutathione (GSH) level of AAILE + DMBA/TPA group. Enhanced LPO level was observed in the hepatic tissue of DMBA/TPA and AAILE + DMBA/TPA groups when compared to control group. However, no alteration was observed in their hepatic GSH levels. The micronuclei score in hepatic tissue did not exhibit significant inter-group differences. The results of the present study suggest that apart from skin, liver may be affected during DMBA/TPA-induced skin tumorigenesis. AAILE treatment has the ability to modulate these changes potentially influencing the process of tumor formation. These findings seem to be important to carcinogenesis and its intervention with anti-cancer agents.

Phorbol 12-Myristate 13-Acetate Enhances Long-Term Potentiation in the Hippocampus through Activation of Protein Kinase Cdelta and epsilon

Many intracellular proteins and signaling cascades contribute to the sensitivity of N-methyl-D-aspartate receptors (NMDARs). One such putative contributor is the serine/threonine kinase, protein kinase C (PKC). Activation of PKC by phorbol 12-myristate 13-acetate (PMA) causes activation of extracellular signal-regulated kinase (ERK) and promotes the formation of new spines in cultured hippocampal neurons. The purpose of this study was to examine which PKC isoforms are responsible for the PMA-induced augmentation of long-term potentiation (LTP) in the CA1 stratum radiatum of the hippocampus in vitro and verify that this facilitation requires NMDAR activation. We found that PMA enhanced the induction of LTP by a single episode of theta-burst stimulation in a concentration-dependent manner without affecting to magnitude of baseline field excitatory postsynaptic potentials. Facilitation of LTP by PMA (200 nM) was blocked by the nonspecific PKC inhibitor, Ro 31-8220 (10microM); the selective PKCdelta inhibitor, rottlerin (1microM); and the PKCepsilon inhibitor, TAT-epsilonV1-2 peptide (500 nM). Moreover, the NMDAR blocker DL-APV (50microM) prevented enhancement of LTP by PMA. Our results suggest that PMA contributes to synaptic plasticity in the nervous system via activation of PKCdelta and/or PKCepsilon, and confirm that NMDAR activity is required for this effect.

Influence of different T cell activators on the expression of Foxp3 in bronchial asthma / 中华实用儿科临床杂志

Objective To explore the influence of different T cell activators on the expression of Foxp3 in bronchial asthma.Met-hods Peripheral blood monouclear(PBMCs) were isolated from the 30 children suffering from mild persistent asthma allergic to house dust mite (HDM).PBMCs of those children were divided into 2 groups.One group was stimulated with HDM extracts (HDM group) and the other was stimulated with phorbol-12-myristate-13-acetate (PMA) extracts (PMA group).After 48 hours of in vitro stimulation with HDM extracts or PMA extracts,the levels of CD4 + CD25 + T cells,CD4 + CD25 + Foxp3 + T cells and CD4 + CD25 + Foxp3 + IL-10 + T cells were measured by flow cytometry.Results The levels of CD4 + CD25 + T cells significantly decreased in the HDM group[(4.59 ± 1.10) ％] compared with those in the PMA group[(41.24 ±7.95)％] (P ＜0.0001).The levels of CD4+ CD25 + Foxp3 + T cells significantly decreased in the HDM group [(18.08 ± 1.82) ％] compared with those in the PMA group [(90.26 ± 3.63) ％] (P ＜ 0.0001).The levels of CD4 + CD25 + Foxp3 + IL-10 + T cells had no statistical significance in the HDM group [(37.62 ± 3.23) ％] compared with those in the PMA group [(38.33-± 3.95) ％] (P ＞ 0.05).Conclusion Nonspecific T cell activators can increase the expression of Foxp3 in bronchial asthma.

Phorbol 12-Myristate 13-Acetate Induces MUC16 Expression via PKCdelta and p38 in Human Airway Epithelial Cells

OBJECTIVES: Phorbol 12-myristate 13-acetate (PMA) is widely used as a protein kinase C (PKC) activator, PKC is involved in the secretion of mucins. MUC16, one of the membrane-bound mucins, is produced in human airway epithelial cells. However, the effect and signaling pathway of PMA on MUC16 expression in human airway epithelial cells has not been reported. Therefore, the effect and brief signaling pathway of PMA on MUC16 expression were investigated in human airway epithelial cells in this study. METHODS: In the mucin-producing human NCI-H292 airway epithelial cells and the primary cultures of normal nasal epithelial cells, the effect and signaling pathway of PMA on MUC16 expression were investigated using reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, enzyme immunoassay, and immunoblot analysis with several specific inhibitors and small interfering RNA (siRNA) for p38 mitogen-activated protein kinase (MAPK). RESULTS: PMA increased MUC16 expression, and activated phosphorylation of p38 MAPK. However, it did not activate phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). SB203580 (p38 MAPK inhibitor) inhibited PMA-induced MUC16 expression, while U0126 (ERK1/2 inhibitor) did not. In addition, the knockdown of p38 MAPK by p38 MAPK siRNA significantly blocked PMA-induced MUC16 mRNA expression. Rottlerin (PKCdelta inhibitor) inhibited PMA-induced MUC16 expression, and also inhibited the phosphorylation of activated p38 MAPK by PMA. CONCLUSION: These results show for the first time that PMA-induced MUC16 expression is regulated by activation of the PKCdelta and p38 MAPK signaling pathway in human airway epithelial cells.

EFFECT OF MAXIMAL EXERCISE ON NADPH OXIDASE ACTIVITY OF NEUTROPHILS AND RELATION TO SUPEROXIDE-GENERATING ACTIVITY BY CELL-SYSTEM IN HUMAN PERIPHERAL BLOOD / 体力科学

The purpose of this study was to examine reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, - a generated superoxide - of neutrophils in human peripheral blood after maximal exercise. Ten healthy male college students (20.2 ± 0.4 yr, 170.5 ± 1.3 cm, 62.8 ± 1.9 kg) participated after giving written informed consent. They performed an incremental exercise to volitional exhaustion using a bicycle ergometer. Peripheral blood was collected before exercise (Pre), just after exercise (Post) and 1-hour after exercise (Post-1h). Phorbol 12-myristate 13-acetate (PMA)-stimulated and opsonaized zymosan (OZ)-stimulated superoxide-generating activity of neutrophils was measured by the cytochrome c reduction assay. NADPH oxidase activity was measured by a cell-free system. NADPH oxidase activity significantly decreased in Post-1h compared with Pre and Post. A similar tendency was seen in PMA-stimulated activity, but not in OZ-stimulated activity. A strong positive relationship between NADPH oxidase activity and PMA-stimulated activity was found in Pre and this relationship attenuated after exercise. NADPH oxidase activity was not related to OZ-stimulated activity at any time points. We concluded that NADPH oxidase activity decreased after exhaustive maximal exercise in human peripheral neutrophils, and suggest that PMA-stimulated activity, relatively - speaking, reflects NADPH oxidase activity; but OZ-stimulated activity is independent of NADPH oxidase activity.

Effect of the monocyte locomotion inhibitory factor (MLIF) produced by E. histolityca on cytokines and chemokine receptors in T CD4+ lymphocytes

Entamoeba histolytica produces Monocyte Locomotion Inhibitory Factor (MLIF), which may contribute to the delayed inflammation observed in amoebic hepatic abscesses. Leukocytes are affected through the modulation of cytokine expression and/or production. We evaluated the effects of MLIF on the activation and production of intracellular cytokines in human CD4+ T lymphocytes by flow cytometry. Cells were stimulated for 24 h with PMA, MLIF, or PMA+MLIF. Cellular activation was measured using anti-CD69. Th1/Th2 production was studied by the expression of intracellular cytokines and cytokine/chemokine receptors. MLIF increased CD69 and induced the over-expression of the IL-l©¬, IFN-¥ã, IL-2, IL-4, and IL-10 intracellular cytokines; PMA+MLIF inhibited Th1 cytokine (IFN-¥ã) and increased Th2 cytokines (IL-4 and IL-10). The co-expression of the cytokine and chemokine receptors IFN-¥ã/CCR5 and IL-1©¬/CCR5 was inhibited by PMA+MLIF and Th2 co-expression was increased. MLIF effects varied depending on the conditions. MLIF alone activated the Th1 and Th2 cytokines and cytokine/receptor expression; however, PMA+MLIF increased the expression of Th2 but inhibited it in Th1.

Effect of phorbol-12-myristate-13-acetate on cyclooxygenase-2 expression in human HaCaT keratinocytes / 中华皮肤科杂志

Objective To investigate the effect of phorbol-12-myristate-13-acetate(PMA)on cyclooxygenase-2(COX-2) mRNA and protein expression in cultured human HaCaT keratinocytes,and the mechanism for cytotoxity of PMA against keratinocytes.MethodsRT-PCR and Westem blot were utilized to detect the expression of COX-2 mRNA and protein in cultured HaCaT ells at 24 hours after the treatment with various concentrations of PMA (0.1,1.0,10 mg/L).ResultsWithout any treatment,there was no or a weak expression df COX-2 mRNA and protein in HaCaT cells;incubation witll PMA resulted in the induction of the expression of COX-2 in HaCaT cells.The expression levels of COX-2 mRNA and protein in 10 mg/L PMA-pretreated HaCaT cells were significantly higher than those in 1.0 mg/L PMA-pretreated HaCaT cells,which was in turn higher than that in 0.1 mg/L PMA-pretreated cells and untreated cells;the difrerence was statistically significant (all P＜0.01).Conclusion These results suggest that PMA may be involved in keratinocyte tumorigenesis by upregulating he expression of COX-2 as well as synthesis and release of prostaglandin in keratinocytes.