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We combined pyropheophorbide-a methyl ester, photodynamic therapy (MPPa-PDT), 670 ± 10 nm, 4 mW/cm2, with herpes simplex virus type 1 thymidine kinase/ganciclovir (HSV1-TK/GCV) to improve the therapeutic effect. We built HSV1-TK expression vector GV230-TK and we observed a bright green fluorescence under fluorescence microscope. It indicated the recombinant plasmid was transfected into PC-3M prostate cancer cells successfully. As the abundant glucose-regulated protein 78 (GRP78) promoter in PC-3M cells can cause active expression of HSV1-TK, cell protein was collected for western blot to determine the expression of HSV1-TK. In CCK-8 assay (n = 6), the cell survival rate of combined treatment group was about 10%, less than that of pure MPPa-PDT group (23%) and pure HSV1-TK/GCV group (35%) (t test, P

The aim of this study was to establish a system for high expression of Aspergillus niger glucose oxidase (GOD) with glyceraldehyde-3-phosphate dehydrogenase gene promoter (pGAP) in Pichia SMD1168. The GOD gene from A. niger accc30161 was inserted into pGAPZαA plasmid carrying a pGAP promoter and was expressed in yeast Pichia pastoris. The expression vector, pGAPZαA-GOD, was validated by colony polymerase chain reaction (PCR), agarose gel electrophoresis, restriction enzyme analysis and sequencing methods. The pGAPZαA-GOD vector was transformed into yeast P. pastoris SMD1168 by electroporation and the positive strain was validated by PCR. The expression and enzyme activity of the recombinant GOD were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and enzymatic detection. The recombinant GOD in yeast was purified by ion exchange chromatography, and its biochemical properties (dynamics, thermal and pH stability) were analysed. The obtained results showed that the expression vector, pGAPZαA-GOD, was successfully constructed to express A. niger accc30161 GOD protein. After transformation, the pGAPZαA-GOD DNA fragment had been integrated into the P. pastoris SMD1168-GOD genome. High expression of GOD was achieved in SMD1168-GOD, and the enzyme activity of GOD reached 107.18 U/mL in the supernatant of the culture medium at 30 °C and pH 6. The activity of recombinant GOD protein in yeast was 1.35-fold higher than the commercial A. niger GOD, and its affinity to glucose, thermal stability and pH stability are similar to commercial GOD. Using pGAP promoter, A. niger GOD protein is efficiently expressed in recombinant P. pastoris SMD1168-GOD with defective protease.

Objectives Cellular senescence is a state of irreversible growth arrest induced either by telomere shortening (replicative senescence) or stress. The bronchial epithelial cell is often injured by inhaled toxic substances, such as cigarette smoke. In the present study, we investigated whether exposure to cigarette smoke extract (CSE) induces senescence of bronchial epithelial cells; and Cordyceps sinensis mechanism of inhibition of CSE-induced cellular senescence.

INTRODUCTION This study aims to compare the antioxidant abilities of cervical squamous carcinoma cells and cervical adenocarcinoma cells and to study the related mechanisms. MATERIAL AND METHODS Cervical squamous carcinoma and adenocarcinoma cells were treated with H2O2. Cell proliferation was determined with the MTT assay. The reactive oxygen species (ROS) level was detected by the 2',7'-dichlorofluorescein-diacetate (DCFH-DA) method. The 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) method was performed to measure intracellular concentrations of reduced glutathione (GSH) and oxidized glutathione (GSSG). The nitrite formation method, the molybdate colorimetric method, and the DTNB colorimetric method were used to determine activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), respectively. RESULTS Compared with untreated control cells, cell proliferation of cervical squamous carcinoma cells and cervical adenocarcinoma cells was significantly inhibited by H2O2 treatment (p < 0.05). Reactive oxygen species levels and GSSG levels were significantly increased (p < 0.01), whereas GSH levels were significantly decreased (p < 0.05 or 0.01) in both cells after H2O2 treatment. Thus the ratio of GSH/GSSG was significantly decreased by H2O2 treatment in both cells (p < 0.01). In addition, H2O2 treatment significantly increased activities of SOD, CAT, and GPx in both cells (p < 0.05 or 0.01). Furthermore, the above-mentioned changes induced by H2O2 treatment were more dramatic in cervical squamous carcinoma cells. CONCLUSIONS The antioxidant ability of cervical squamous carcinoma cells is lower than that of cervical adenocarcinoma cells, which may be related to the increased ROS levels in cervical squamous carcinoma cells induced by H2O2 treatments.

The aim of our study was to elucidate the function and signaling pathway of found in inflammatory zone 1 (FIZZ1) in airway remodeling in asthma. We used a mice model sensitized and challenged by ovalbumin (OVA) to evaluate the expression of FIZZ1, type I collagen, and fibronectin-1 in the airway in asthma. To investigate the signaling pathway regulated by FIZZ1, we treated a cultured murine lung epithelium cell-12 (MLE-12) with FIZZ1 recombination protein, silenced the expression of FIZZ1 with FIZZ1-shRNA in vitro, and then detected phosphorylated phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and expression of type I collagen and fibronectin-1 (FN-1) by Western blotting. In addition, we increased the expression of PTEN by PTEN plasmid transfection then detected the expression of type I collagen and fibronectin-1 in MLE-12 by Western blot analysis and immunofluorescence cytochemistry technology, respectively. First, the expression of FIZZ1, type I collagen, and fibronectin-1 was significantly elevated in the lungs of OVA-challenged mice compared with saline-treated control animals. Secondly, the phosphorylation of PTEN was decreased in MLE-12 treated with FIZZ1 recombination protein in vitro. On the contrary, the phosphorylation of PTEN was increased in MLE-12 cells transfected with FIZZ1-shRNA. Thirdly, results of the Western blot analysis and immunofluorescence cytochemistry showed that expression of type I collagen and fibronectin-1 was increased in cells treated with FIZZ1 recombination protein, while the levels of type I collagen and fibronectin-1 were significantly decreased in cells transfected with PTEN plasmid. FIZZ1 may be a critical cytokine in airway remodeling in asthma. This study indicates that targeting FIZZ1 and/or PTEN may be a new therapeutic strategy for asthma.

MAPK-activating death domain protein (MADD) binds to the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor and acts as a key downstream mediator in the TRAIL-induced apoptosis pathway. The aim of this study was to evaluate the expression of MADD in normal human and adenocarcinoma tissues of the lungs and its influence on proliferation and apoptosis of A549 human lung adenocarcinoma cells. Immunohistochemistry was carried out to detect the expression of MADD in normal and tumor tissues of the lungs. Expression of the MADD gene in A549 cells was measured by reverse transcription-polymerase chain reaction. A549 cells were transfected with plasmids carrying the DNA fragment encoding MADD and lentiviral vectors used for RNA interference, respectively. MADD expression in the transfected A549 cells was determined by western blotting. Proliferation and apoptosis were detected using MTT assay and flow cytometry, respectively. It was found that non-small cell lung cancer tissues expressed MADD at higher levels compared to normal lung tissues, and the level of MADD in lung adenocarcinoma was higher compared to that in lung squamous cell carcinoma. MADD was expressed in A549 cells. Both introduction of the DNA fragment encoding MADD and RNA interference targeting MADD effectively altered levels of MADD in the A549 cells. Overexpression of MADD in the A549 cells inhibited apoptosis and increased survival whereas abrogation of MADD promoted apoptosis and reduced cell proliferation. These results suggest that MADD may be a potential therapeutic target for lung adenocarcinoma therapy involving the TRAIL-induced apoptosis pathway.

Macroautophagy (autophagy) is crucial for cell survival during starvation and plays important roles in human diseases. It is a highly conserved intracellular degradation system in eukaryotes for removal and recycling of cytoplasmic components including damaged proteins and organelles to obtain energy. The relationship between cancer and autophagy has been extensively studied in recent years. In cancer and cancer therapy, autophagy acts as a double-edged sword. Photodynamic therapy (PDT) is a kind of tumor therapy applied with a tumor-localizing photosensitizing agent which is followed by activation with the light of a specific wavelength. How much is autophagy involved in photodynamic therapy? The work in this area is still limited. Keywords : Autophagy, Photodynamic therapy, Apoptosis, Cancer

This study aimed to identify the role and regulation of thymic stromal lymphopoietin (TSLP) in asthmatic airway remodelling. To identify the expression of TSLP, α smooth muscle actin (α-SMA) and collagen I in bronchial tissues, bronchial biopsy specimens were collected from patients with asthma and healthy controls and stained with specific antibodies, respectively. To characterize the signalling pathways regulated by TSLP, we silenced or overexpressed TSLP in human lung fibroblast (HLF-1) cells by shRNA approaches or transfection and detected the expression of TSLP receptor (TSLPR) by enzyme-linked immunosorbent assay and Western blot analysis. In TSLP signalling pathway, the protein expression of total signal transducer and activator of transcription 3 (STAT3), STAT5, the phosphorylation of STAT3 (pSTAT3) and STAT5 (pSTAT5), TSLP, α-SMA and collagen I were also detected by Western blotting. In addition, the α-SMA, collagen I and mRNA expression were determined by real-time reverse-transcription. To further confirm the TSLP-STAT3 signalling pathway in HLF-1 cells, we inhibited STAT3 activity by targeted small molecules and then detected TSLP-induced expression of α-SMA and collagen I in both mRNA and protein levels by quantitative real-time reverse-transcription and Western blotting, respectively. First, overexpression of TSLP, α-SMA and collagen I was detected in epithelium collected from patients with asthma. Second, STAT3 activity and the expression of α-SMA and collagen I were controlled, regulated by TSLP. Specifically, the pSTAT3, α-SMA and collagen I were induced by the introduction of TSLP in HLF-1 cells, and the repression of α-SMA and collagen I was detected after TSLP silencing. Third, no changes of pSTAT5 were found in the presence of the STAT3 inhibitor, and TSLP-induced α-SMA and collagen I upregulation is in a STAT3 dependent manner. If we inhibit STAT3 activity by STAT3 targeted small molecules, TSLP-induced α-SMA and collagen I upregulation cannot be detected. The functions of TSLP in asthmatic airway remodelling were performed through STAT3 signalling pathway.

Asthma is an epithelial disorder in which T helper 2 (Th2)-type inflammation has a prominent role. Recent studies indicated that a cytokine, thymic stromal lymphopoietin (TSLP), is essential for the development of antigen-induced asthma. The authors used ovalbumin (OVA) sensitization and challenge to induce a murine asthmatic model. The model was confirmed by airway hyperresponsiveness, serum levels of total and OVA-specific immunoglobulin (IgE), histological analysis of lung tissues. The authors found that expression of TSLP was significantly increased in both mRNA and protein levels in mice lungs treated with OVA. The expression of CD40, CD80, and CD86 in bronchoalveolar lavage fluid (BALF) was increased in mice with OVA. Tight correlation between TSLP mRNA and interleukin (IL)-4, IL-5, and IL-13 in BALF was identified. Furthermore, treating mice with TSLP-neutralizing antibody reduced the expression of TSLP mRNA of lungs, CD40, CD80, and CD86 on dendritic cells, and IL-4, IL-5, and IL-13 in the OVA group. This study indicates that TSLP is increased in the airway epithelium in mice treated with OVA. In the lung inflammation model, TSLP activates dendritic cells (DCs) via up-regulation of CD40, CD80, and CD86, then induces the differentiation of prime naive CD4(+) T cells to become proinflammatory Th2 cells. Blocking TSLP is capable of inhibiting the production of Th2 cytokines, thus presents a promising strategy for the treatment of asthma.

The aim of this study was to elucidate the role of Found in Inflammatory Zone 1 (FIZZ1, also known as RELM-alpha or resistin-like molecule-alpha) in airway remodeling in asthma. We used a rat model of ovalbumin (OVA) sensitization and challenge to induce lung inflammation and remodeling. Expression of alpha -SMA in the lungs of OVA-treated rats was significantly elevated in the peribronchial regions compared with control saline-treated animals. Expression of FIZZ1 mRNA in alveolar epithelial type II cells (AECII) isolated from OVA-treated animals was higher than in control animals. Forced expression of recombinant FIZZ1 in rat-1 lung fibroblast cell line enhanced production of collagen type I and alpha -SMA compared with control transfected cells. These results suggest that FIZZ1 can induce fibroblasts to express markers of myofibroblast differentiation such as alpha -SMA and collagen type I, which are characteristic of early stages of airway remodeling seen in asthma.

We compared relative copper-binding capacities of cellular components and determined the substance that dominantly sequesters excess copper incorporated into bakers' yeast cells before the copper homeostasis systems start to operate. The fluorescent chelator calcein, whose fluorescence is quenched by its binding with copper, was used for estimation of the copper-binding capacities. The copper-binding capacity was assessed by the degree of competition of a sample against calcein for copper. The result indicated that GSH and a whole of the other low-molecular-weight substances had comparable copper-binding capacities, which were a little higher than that of cytosolic proteins as a whole, when compared at their relative cellular concentrations. However, a centrifugal ultrafiltration study showed that essentially all the copper ions incorporated in cells after treatment with 50 μM CuCl2 for 5 min were not filterable, indicating their binding to high-molecular-weight substances, namely cytosolic proteins. In the direct fluorescence measurement with calcein-loaded cells, no labile copper was detected in either wild-type yeast or GSH-deficient mutant upon treatment with 50 μM CuCl2, supporting the notion of the copper buffering by the cytosolic proteins under the in vivo conditions. This property of the proteins may not only play a part in cell's resistance against copper but also it may necessitate the role of copper chaperones in the intracellular trafficking of copper to target proteins.

The aim of our study was to elucidate the function and signaling pathway of found in inflammatory zone 1 (FIZZ1) in airway remodeling in asthma. We used a mice model sensitized and challenged by ovalbumin (OVA) to evaluate the expression of FIZZ1, type I collagen, and fibronectin-1 in the airway in asthma. To investigate the signaling pathway regulated by FIZZ1, we treated a cultured murine lung epithelium cell-12 (MLE-12) with FIZZ1 recombination protein, silenced the expression of FIZZ1 with FIZZ1-shRNA in vitro, and then detected phosphorylated phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and expression of type I collagen and fibronectin-1 (FN-1) by Western blotting. In addition, we increased the expression of PTEN by PTEN plasmid transfection then detected the expression of type I collagen and fibronectin-1 in MLE-12 by Western blot analysis and immunofluorescence cytochemistry technology, respectively. First, the expression of FIZZ1, type I collagen, and fibronectin-1 was significantly elevated in the lungs of OVA-challenged mice compared with saline-treated control animals. Secondly, the phosphorylation of PTEN was decreased in MLE-12 treated with FIZZ1 recombination protein in vitro. On the contrary, the phosphorylation of PTEN was increased in MLE-12 cells transfected with FIZZ1-shRNA. Thirdly, results of the Western blot analysis and immunofluorescence cytochemistry showed that expression of type I collagen and fibronectin-1 was increased in cells treated with FIZZ1 recombination protein, while the levels of type I collagen and fibronectin-1 were significantly decreased in cells transfected with PTEN plasmid. FIZZ1 may be a critical cytokine in airway remodeling in asthma. This study indicates that targeting FIZZ1 and/or PTEN may be a new therapeutic strategy for asthma.

This study is to establish a paclitaxel (PTX)-resistant human cervical carcinoma HeLa cell line (HeLa/PTX) and to investigate its redox characteristics and the expression of taxol resistance gene 1 (Txr1). HeLa cells were treated with PTX and effects of PTX on cell proliferation were detected through cell counting and the MTT assay. Levels of cellular reactive oxygen species (ROS), reduced glutathione (GSH), and oxidized glutathione (GSSG) as well as the ratio of GSH to GSSG were measured by the 2,7-difluorescein diacetate (DCFH-DA) method and the 5,5'dithiobis(2-nitrobenzoic acid) (DTNB) method. Activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were determined by the nitrite formation method, the molybdate colorimetric method, and the DTNB colorimetric method, respectively. The level of Txr1 mRNA was determined by real-time PCR. Compared with the regular HeLa cells, HeLa/PTX cells were larger in size and had more cytoplasmic granules. The population doubling time for HeLa/PTX cells was 1.32 times of that of HeLa cells (P0.01). HeLa/PTX cells showed stronger resistance to PTX than HeLa cells with a resistance index of 122.69. HeLa/PTX cells had higher levels of ROS (P0.01) and Txr1 mRNA (P0.01), lower level of GSH (P0.05), and lower activities of SOD (P0.01) and GPx (P0.05) than HeLa cells. HeLa/PTX cells, with higher levels of ROS and Txr1 mRNA expression, are more resistant to PTX than HeLa cells.

Found in inflammatory zone 1 (FIZZ1) plays a vital role in pulmonary inflammation and angiogenesis. In addition, FIZZ1 plays a role in the early stages of airway remodeling in asthma by increasing the expression of α smooth muscle actin (α-SMA) and type I collagen. However, the role of FIZZ1 in the airway remodeling of asthma remains unclear. In the present study, FIZZ1 was identified to be upregulated in ovalbumin (OVA)-induced asthmatic mice, along with phosphorylated protein kinase B (Akt). Following FIZZ1 recombinant protein co-culture in the murine lung epithelial-cell line, Akt phosphorylation was upregulated, however, following transfection with FIZZ1-small hairpin RNA, the phosphorylation levels were decreased. The variation in α-SMA and type I collagen expression levels was consistent with the Akt phosphorylation levels. Intratracheal administration of LY294002 and Akt inhibitor IV to the asthmatic mice was capable of reducing airway inflammation, downregulating the expression of α-SMA, type I collagen and fibronectin-1 and increasing the expression of E-cadherin. In conclusion, the present study demonstrated that FIZZ1 promoted airway remodeling in asthma via the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. Blocking the PI3K/Akt signaling pathway may attenuate the early stages of airway remodeling induced by OVA by regulating the abnormal process of epithelial-mesenchymal transition.

This study aimed to compare the efficacy and safety of thrombolytic and anticoagulant therapy for acute submassive pulmonary embolism (PE). A retrospective evaluation was performed on 25 consecutive inpatients with acute submassive PE treated by thrombolytic therapy and 25 earlier consecutive inpatients with acute submassive PE treated by anticoagulant therapy. No statistically significant difference in clinical curative effect was identified between the thrombolysis and anticoagulation groups (P>0.05). Following 24 h of therapy, the improvement rates of dyspnea and revascularization in the thrombolysis group achieved statistical significance compared with those of the anticoagulation group (P

Photodynamic therapy (PDT) is a medical treatment using a photosensitizing agent and light source to treat cancers. Pyropheophorbidea methyl ester (MPPa), a derivative of chlorophyll, is a novel potent photosensitizer. To learn more about this photosensitizer, we examined the cell cycle arrest in MDA-MB-231. Cell cycle and apoptosis were measured by flow cytometer. Checkpoints of the cell cycle were measured by western blot. In this study, we found that the expression of Cyclin D1 was obviously decreased, while the expression of Chk2 and P21 was increased after PDT treatment. This study showed that MPPa-PDT affected the checkpoints of the cell cycle and led the cells to apoptosis.

To study the activation of macrophage and upregulation of costimulatory molecule of CD40 in lipopolysaccharide- (LPS-) induced acute lung injury (ALI) model, and to investigate the pathogenecy of ALI, mice were randomly divided into two groups. ALI model was created by injecting 0.2 mg/kg LPS in phosphate saline (PBS) in trachea. The pathologic changes of mice lungs were observed by HE staining at 24 and 48 hours after LPS treatment, then the alveolar septum damage, abnormal contraction, alveolar space hyperemia, and neutrophils or other inflammatory cells infiltration in the LPS group, but not in the control group, were observed. The expression of CD40 mRNA and CD40 protein molecules were higher in LPS group as compared to the control group by Northern blot and flow cytometry, respectively. Expression of Toll-like receptor-4 (TLR4) in activated macrophage (AMPhi) was higher in LPS group as compared to the control group by RT-PCR. The activation of NF-kappaB binding to NF-kappaB consensus oligos increased in LPS group by EMSA in macrophage. The concentrations of TNF-alpha, MIP-2, and IL-1beta cytokines from bronchoalveolar lavage fluid (BALF) were increased significantly in LPS group as compared to the control group by ELISA. The activation of AM and upregulation of costimulatory molecule CD40 induced all kinds of inflammatory cytokines releasing, then led to ALI. Therefore, both of them played vital role in the process of development of ALI.

It has been amply documented that L-ascorbic acid added to the medium of a cell culture increases oxidative damage, and this effect of L-ascorbic acid has been ascribed to the generation of reactive oxygen intermediates in the medium during its auto-oxidation. We have here questioned whether such an effect is exerted inside the cell as well, and if so, what its mechanism is. To assess thiol oxidation in the cell, we manipulated CHO cells so that they could express bacterial alkaline phosphatase in the cytoplasm. Alkaline phosphatase activity, which requires the formation of intramolecular disulfide bridges, was shown to appear when the cells were exposed to H2O2. This H2O2-induced activity increased more than 1.5 fold when L-ascorbic acid had been loaded in the cells by incubation with L-ascorbic acid-2-O-phosphate. Similar enhancing effects were also observed by assessing oxidation of glutathione, formation of protein carbonyls, and generation of reactive oxygen intermediates. Interestingly, the effects by the L-ascorbic acid-2-O-phosphate treatment were totally suppressed by addition of the membrane-permeable chelator deferoxamine to the medium, indicating the involvement of iron ions. Because the apoprotein of conalbumin, which binds iron ions with a high affinity, had no effect and because the same deferoxamine effect was observed with the cells incubated in balanced salt solution with no metal salts added, it was concluded that L-ascorbic acid acts as a pro-oxidant within the cell suffering oxidative stress, and that this effect is elicited through increased redox-cycling of iron in combination with L-ascorbic acid.

Prion protein consists of an N-terminal domain containing a series of octapeptide repeats with the consensus sequence PHGGGWGQ and a C-terminal domain composed of three α-helices and two short β-strands. Several studies have shown that the N-terminal domain binds five Cu2+ ions. In the present study, we have investigated copper-catalysed oxidation of a recombinant mouse prion protein, PrP23–231. The copper-loaded PrP23–231 was found to be carbonylated by incubation with dopamine. Besides the formation of carbonyls, a cross-linked species with the dimeric size and C-terminally truncated species were generated. These reactions were retarded in the presence of Cu+- and Cu2+-specific copper chelators, catalase, and SOD (superoxide dismutase), but not in the presence of various bivalent metal ions. Together, these results indicate that the copper bound to prion protein undergoes catalytic cycling in the presence of catecholamines and causes the oxidation of the protein.