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Erlotinib (Tarceva) is a selective epidermal growth factor receptor tyrosine kinase inhibitor in the treatment of human non–small cell lung cancer (NSCLC). In this study, we investigated the roles of ERK1/2 and AKT signaling pathways in regulating Rad51 expression and cytotoxic effects in different NSCLC cell lines treated with erlotinib. Erlotinib decreased cellular levels of phosphorylated ERK1/2, phosphorylated AKT, Rad51 protein, and mRNA in erlotinib-sensitive H1650, A549, and H1869 cells, leading to cell death via apoptosis, but these results were not seen in erlotinib-resistant H520 and H1703 cells. Erlotinib decreased Rad51 protein levels by enhancing Rad51 mRNA and protein instability. Enforced expression of constitutively active MKK1 or AKT vectors could restore Rad51 protein levels, which were inhibited by erlotinib, and decrease erlotinib-induced cytotoxicity. Knocking down endogenous Rad51 expression by si-Rad51 RNA transfection significantly enhanced erlotinib-induced cytotoxicity. In contrast, overexpression of Rad51 by transfection with Rad51 vector could protect the cells from cytotoxic effects induced by erlotinib. Blocking the activations of ERK1/2 and AKT by MKK1/2 inhibitor (U0126) and phosphoinositide 3-kinase inhibitor (wortmannin) suppressed the expression of Rad51 and enhanced the erlotinib-induced cell death in erlotinib-resistant cells. In conclusion, suppression of Rad51 may be a novel therapeutic modality in overcoming drug resistance of erlotinib in NSCLC. (Mol Cancer Res 2009;7(8):1378–89)

Supplementary Data from Roles of MKK1/2-ERK1/2 and Phosphoinositide 3-Kinase–AKT Signaling Pathways in Erlotinib-Induced Rad51 Suppression and Cytotoxicity in Human Non–Small Cell Lung Cancer Cells

Supplementary Fig. S2 from Role of repair protein Rad51 in regulating the response to gefitinib in human non-small cell lung cancer cells

Gefitinib (Iressa, ZD1839) is a selective epidermal growth factor receptor tyrosine kinase inhibitor that can block growth factor-mediated cell proliferation and extracellular signal-regulated kinases 1/2 (ERK1/2) activation. High-level Rad51 expression has been reported in chemoresistant or radioresistant carcinomas. In this study, we examined the role of Rad51 in regulating the response to gefitinib among different human lung cancer cell lines. The H520 line (human squamous cell carcinoma) was less sensitive to gefitinib compared with the H1650 (human adenocarcinoma) or A549 (human bronchioloalveolar carcinoma) lines. In H1650 and A549 cells but not in H520 cells, gefitinib decreased cellular levels of phospho-ERK1/2 and Rad51 protein and message levels. Moreover, gefitinib decreased Rad51 protein levels by enhancing Rad51 protein instability through 26S proteasome-mediated degradation. Inhibition of endogenous Rad51 levels by si-Rad51 RNA transfection significantly enhanced gefitinib-induced cytotoxicity. In contrast, transfection with constitutively active MKK1 vector could restore both Rad51 protein levels and cell survival inhibited by gefitinib. The MKK1/2-ERK1/2 signaling pathway constitutes the upstream signaling for maintaining Rad51 message and protein levels. Rad51 protein can protect lung cancer cells from cytotoxic effects induced by gefitinib. Suppression of Rad51 may be a novel lung cancer therapeutic modality to overcome drug resistance to gefitinib. [Mol Cancer Ther 2008;7(11):3632–41]

Funder: BBSRC BrightStar Studentship, BACKGROUND: Osteoarthritis (OA) in the horse is an economic and welfare issue and there are no current disease modifying drugs available. Stem cells have been suggested as a therapeutic intervention for OA, originally on the basis of their regenerative capacity. However, it is hypothesised that mesenchymal stem cells (MSC) exert their effects via paracrine factors including the production of extracellular vesicles that can themselves recapitulate the MSC effects in the joint. OBJECTIVES: To isolate extracellular vesicles from bone marrow MSC and investigate their anti-inflammatory effects on chondrocytes. STUDY DESIGN: An in vitro assessment of the effect of direct culturing extracellular vesicles on artificially inflamed chondrocytes. METHODS: Extracellular vesicles were isolated from bone marrow MSC using differential sequential ultracentrifugation. Vesicles were characterised using electron microscopy, nanoparticle tracing analysis and protein analysis. Vesicle internalisation was carried out via vesicles being pre-stained and co-cultured with equine chondrocytes before analysis using confocal microscopy. The effects of vesicles on artificially inflamed chondrocytes was examined using quantitative PCR. RESULTS: To the best of the authors' knowledge, this is the first study to isolate and characterise extracellular vesicles from equine bone MSC. Vesicles were taken up by autologous chondrocytes and had anti-inflammatory effects on gene expression following chondrocyte exposure to tumour necrosis factor α and Interleukin 1β. MAIN LIMITATIONS: Only three independent biological repeats were performed and the work was done in vitro. CONCLUSION: Extracellular vesicles can be isolated from equine bone marrow MSC; they may be taken up by chondrocytes and have an anti-inflammatory action.

We have designed and synthesized off–on fluorometric probes, 7-(but-3-yn-1-yloxy)-8-(difluoromethyl)-2H-chromen-2-one (AYF) and 7-((4-(tert-butyldiphenylsilyloxy)benzyl)oxy)-8-(difluoromethyl)coumarin (DPF1), by incorporating the concept of biological signal transduction strategy into their molecular design and present their proof-of-concept demonstration for mercury (Hg2+) detection. AYF undergoes a cascade of self-immolative reactions in the presence of Hg2+ and unmasks fluorogenic coumarin 1 and releases two latent fluoride ions. These fluorides initiate the removal of silyl protecting trigger group on DPF1 which directs another cascade of self-immolative reactions. The fluoride ions (amplifiers) are continuously activating the cascade reaction, and, as a result, coumarin 1 progressively accumulates in the reaction medium and the whole process accelerates an amplified signal for Hg2+. For detection of Hg2+, the limit of detection of the two-probe coupling assay (AYF and DPF1) is 0.51 ppb which is more than 2000 times lower than that of AYF alone. Besides, 3-(benzothiazol-2-yl)-4-carbonitrile-7-((4-(tert-butyldiphenylsilyloxy)benzyl)oxy)coumarin (DCC), a long-wavelength probe, was coupled with AYF and DPF1 in order to shift the emission to a higher wavelength region. Negative control studies revealed that the probes undergo controlled and organized sensing pathways as we designed.

Drug resistance is a frequent cause of failure in cancer chemotherapy treatments. In this study, a pair of uterine sarcoma cancer lines, MES-SA, and doxorubicin-resistant partners, MES-SA/DxR-2μM cells and MES-SA/DxR-8μM cells, as a model system to investigate resistance-dependent proteome alterations and to identify potential therapeutic targets. We used two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to perform this research and the results revealed that doxorubicin-resistance altered the expression of 208 proteins in which 129 identified proteins showed dose-dependent manners in response to the levels of resistance. Further studies have used RNA interference, H2A.X phosphorylation assay, cell viability analysis, and analysis of apoptosis against reticulocalbin-1 (RCN1) proteins, to prove its potency on the formation of doxorubicin resistance as well as the attenuation of doxorubicin-associated DNA double strand breakage. To sum up, our results provide useful diagnostic markers and therapeutic candidates such as RCN1 for the treatment of doxorubicin-resistant uterine cancer.

Neuroendocrine cervical cancer is an aggressive but rare form of cervical cancer. The majority of neuroendocrine cervical cancer patients present with advanced-stage diseases. However, the limited numbers of neuroendocrine tumor markers are insufficient for clinical purposes. Thus, we used a proteomic approach combining lysine labeling 2D-DIGE and MALDI-TOF MS to investigate the biomarkers for neuroendocrine cervical cancer. By analyzing the global proteome alteration between the neuroendocrine cervical cancer line (HM-1) and non-neuroendocrine cervical cancer lines (CaSki cells, ME-180 cells, and Hela cells), we identified 82 proteins exhibiting marked changes between HM-1 and CaSki cells, and between ME-180 and Hela cells. Several proteins involved in protein folding, cytoskeleton, transcription control, signal transduction, glycolysis, and redox regulation exhibited significant changes in abundance. Proteomic and immunoblot analyses indicated respective 49.88-fold and 25-fold increased levels of transgelin in HM-1 cells compared with that in other non-neuroendocrine cervical cancer cell lines, implying that transgelin is a biomarker for neuroendocrine cervical cancer. In summary, we used a comprehensive neuroendocrine/non-neuroendocrine cervical cancer model based proteomic approach for identifying neuroendocrine cervical cancer markers, which might contribute to the prognosis and diagnosis of neuroendocrine cervical cancer.

The nucleus is a key organelle in mammary cells, which is responsible for several cellular functions including cell proliferation, gene expression, and cell survival. In addition, the nucleus is the primary targets of doxorubicin treatment. In the current study, low-abundance nuclear proteins were enriched for proteomic analysis by using a state-of-the-art two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) strategy to compare and identify the nuclear protein profiling changes responsible for the development of doxorubicin resistance in human uterine cancer cells. The results of the nuclear proteomic analysis indicated that more than 2100 protein features were resolved from an equal pooled amount of three purified nuclear proteins and 117 differentially expressed spots were identified. Of these 117 identified proteins, 48 belonged to nuclear proteins and a positive correlation was observed between the expression levels of 32 of these nuclear proteins and an increase in drug resistance. According to our review of relevant research, nuclear proteins such as DNA repair protein XRCC3 (XRCC3) have not been reported to play roles in the formation of doxorubicin resistance. Previous studies have used RNA interference and cell viability analysis to evidence the essential roles of XRCC3 on its potency in the formation of doxorubicin resistance. To sum up, our nuclear proteomic approaches enabled us to identify numerous proteins, including XRCC3, involved in various drug-resistance-forming mechanisms. Our results provide potential diagnostic markers and therapeutic candidates for treating doxorubicin-resistant uterine cancer.

Doxorubicin is an anticancer drug used in a wide range of cancer therapies; however, doxorubicin-induced drug resistance is one of the most serious obstacles of cancer chemotherapy. Recent studies have indicated that reduced oxidative stress levels in cancer cells induce drug resistance. However, the redox-modifications of resistance – associated cellular targets are largely unknown. Thus, the current study employed cysteine-labeling based two-dimensional differential gel electrophoresis (2D-DIGE) combined with MALDI-TOF mass spectrometry (MALDI-TOF MS) to analyze the effect of doxorubicin resistance on redox regulation in uterine cancer and showed 33 spots that were significantly changed in thiol reactivity. These proteins involve cytoskeleton regulation, signal transduction, redox-regulation, glycolysis, and cell-cycle regulation. The current work shows that the redox 2D-DIGE-based proteomic strategy provides a rapid method to study the molecular mechanisms of doxorubicin-induced drug resistance in uterine cancer. The identified targets may be used to further evaluate their roles in drug-resistance formation and for possible diagnostic or therapeutic applications.

Diabetic retinopathy occurs in approximately 25% of patients with type 1 or type 2 diabetes; the disease can cause poor vision and even blindness because high glucose levels weaken retinal capillaries, causing leakage of blood into surrounding areas. We adopted a proteomics-based approach using 2D-DIGE and MALDI-TOF/TOF MS to compare the differential plasma proteome between diabetic retinopathy with significant retinopathy occurrence within 5 years after diagnosis of diabetes, and diabetic non-retinopathy without diagnosed retinopathy for more than 10 years after diagnosis of diabetes. We identified 77 plasma proteins, which represent 28 unique gene products. These proteins mainly have inflammatory response and coagulation roles. Our approach identified several potential diabetic retinopathy biomarkers including afamin and the protein arginine N-methyltransferase 5, which may be associated with the progression and development of diabetes. In conclusion, we report a comprehensive patient-based plasma proteomic approach to the identification of potential plasma biomarkers for diabetic retinopathy screening and detection.

In mammals, sex development is genetically and hormonally regulated. The process starts with the establishment of chromosomal structures (XY or XX), followed by the expression of sex-dependent genes. In order to elucidate the differential protein profiles between male and female amniocytes, a proteomic approach has been performed in this study. Here, we utilized a proteomics-based approach including 2D-DIGE and MALDI-TOF MS analysis to obtain differentially expressed proteins between male and female amniocytes. After resolving protein samples with 2D-DIGE technique, 45 proteins corresponding to 28 unique proteins were differentially expressed between male and female amninocytes from three independent batches of amniotic fluid. Of all of these unique identified spots, five of them (annexin A1, cathepsin D, cytoskeletal 19, protein disulfide-isomerase, and vimentin) exhibited more than 1.5-fold upregulation or downregulation in at least two independent experiments. Importantly, the identified proteins involved in protein degradation and protein folding display upregulated in male amniocytes, implying the differential regulations of protein degradation and protein folding during sex development. In conclusion, the identified differentially expressed proteins may be employed as potential signatures for the sex development. Moreover, the established proteomic platform might further utilize to discover the potential biomarkers for the prenatal genetic disorders in fetus.

This article unveils the development of a paper-based analytical device designed to rapidly detect and clinically diagnose paraquat (PQ) poisoning. Using wax printing technology, we fabricated a PQ detection device by pattering hydrophobic boundaries on paper. This PQ detection device employs a colorimetric sodium dithionite assay or an ascorbic acid assay to indicate the PQ level in a buffer system or in a human serum system in 10 min. In this test, colorimetric changes, blue in color, were observable with the naked eye. By curve fitting models of sodium dithionite and ascorbic acid assays in normal human serum, we evaluated serum PQ levels for five PQ-poisoned patients before hemoperfusion (HP) treatment and one PQ-poisoned patient after HP treatment. As evidenced by similar detection outcomes, the analytical performance of our device can compete with that of the highest clinical standard, i.e., spectrophotometry, with less complicated sample preparation and with more rapid results. Accordingly, we believe that our rapid PQ detection can benefit physicians determining timely treatment strategies for PQ-poisoned patients once they are taken to hospitals, and that this approach will increase survival rates.

The yeast Saccharomyces cerevisiae contains two distinct nuclear glycyl-tRNA synthetase (GlyRS) genes, GRS1 and GRS2. GRS1 is dual functional in that possesses both cytoplasmic and mitochondrial activities, whereas GRS2 is pseudogene-like. GlyRS1 and GlyRS2 are highly similar on the whole but are distinguished by a lysine-rich insertion domain of 44 amino acid residues, present only in GlyRS1. We herein present evidence that whereas the insertion domain is dispensable for the complementary activity of GRS1in vivo, deletion of this domain from GlyRS1 reduced its aminoacylation activity by up to 9-fold. On the other hand, fusion of a constitutive ADH promoter to GRS2 failed to confer a functional phenotype to the gene, but further fusion of ARC1 (a yeast gene encoding a tRNA-binding protein, Arc1p) to this hybrid gene successfully rescued its activity. Most intriguingly, purified GlyRS2 retained a substantial level of aminoacylation activity. Fusion of Arc1p to this enzyme further enhanced its activity and stability. These findings highlight not only the structural integrity of the pseudogene-encoded enzyme but also the necessity of obtaining an auxiliary tRNA-binding domain for functioning of a yeast tRNA synthetase.

Emodin, a tyrosine kinase inhibitor, is a natural anthraquinone derivative found in the roots and rhizomes of numerous plants; it exhibits an anticancer effect on many malignancies. The most important chemotherapeutic agent for patients with advanced non-small cell lung cancer (NSCLC) is a platinum-containing compound such as cisplatin or carboplatin. The molecular mechanism underlying decreased NSCLC cell viability after treatment with emodin and cisplatin is unclear. Therefore, the aim of this study was to assess the cytotoxic effect of combined emodin and cisplatin on NSCLC cell lines and to clarify underlying molecular mechanisms. Exposure of human NSCLC cells to emodin decreased cisplatin-elicited ERK1/2 activation and ERCC1 protein induction by increasing instability of ERCC1 protein. Cisplatin alone did not affect expression of ERCC1 mRNA. However, emodin alone or combined with cisplatin significantly decreased expression of ERCC1 mRNA levels. Enhancement of ERK1/2 activation by transfection with constitutively active MKK1/2 (MKK1/2-CA) vector increased ERCC1 protein levels and protein stability, as well as increasing viability of NSCLC cells treated with emodin and cisplatin. In contrast, blocking ERK1/2 activation by U0126 (an MKK1/2 inhibitor) decreased cisplatin-elicited ERCC1 expression and enhanced cisplatin-induced cytotoxicity. Depletion of endogenous ERCC1 expression by si-ERCC1 RNA transfection significantly enhanced cisplatin's cytotoxic effect. In conclusion, ERCC1 protein protects NSCLC cells from synergistic cytotoxicity induced by emodin and platinum agents. Further investigation of combined emodin and cisplatin may lead to novel therapy in the future for NSCLC through down-regulating expression of ERCC1.

Emodin, a tyrosine kinase inhibitor, is a natural anthraquinone derivative found in the roots and rhizomes of numerous plants. Emodin exhibits anticancer effects against a variety of cancer cells, including lung cancer cells. ERCC1 and Rad51 proteins are essential for nucleotide excision repair and homologous recombination, respectively. Furthermore, ERCC1 and Rad51 overexpression induces resistance to DNA-damaging agents that promote DNA double-strand breaks. Accordingly, the aim of this study was to determine the role of ERCC1 and Rad51 in emodin-mediated cytotoxicity in human non-small cell lung cancer (NSCLC) cells. Both ERCC1 and Rad51 protein levels as well as mRNA levels were decreased in four different NSCLC cell lines after exposure to emodin. These decreases correlated with the inactivation of the MKK1/2-ERK1/2 pathway. Moreover, cellular ERCC1 and Rad51 protein and mRNA levels were specifically inhibited by U0126, a MKK1/2 inhibitor. We found that transient transfection of human NSCLC cells with si-ERCC1 or si-Rad51 RNA and cotreatment with U0126 could enhance emodin-induced cytotoxicity. In contrast, overexpression of constitutively active MKK1/2 vectors (MKK1/2-CA) was shown to significantly recover reduced phospho-ERK1/2, ERCC1, and Rad51 protein levels and to rescue cell viability upon emodin treatment. These results demonstrate that activation of the MKK1/2-ERK1/2 pathway is the upstream signal regulating the expressions of ERCC1 and Rad51, which are suppressed by emodin to induce cytotoxicity in NSCLC cells.

The transformation of a normal cell into a cancer cell has been correlated with alterations in gene regulation and protein expression. To identify altered proteins and link them to the tumorigenesis of breast cancer, we have distinguished normal breast cells (MCF-10A) from noninvasive breast cancer cells (MCF-7) and invasive breast cancer cells (MB-MDA-231) to identify potential breast cancer markers in transformed breast cells. Using the 2D-DIGE and MALDI-TOF MS techniques, we quantified and identified differentially expressed extracellular secreted proteins and total cellular proteins across MCF-7, MB-MDA-231 and MCF-10A. The proteomic analysis of the secreted proteins identified 50 unique differentially expressed proteins from three different media. In addition, 133 unique differentially expressed proteins from total cellular proteins were also identified. Note that 14 of the secreted proteins and 51 of the total cellular proteins have not been previously reported in breast cancer research. Some of these unreported proteins have been examined in other breast cancer cell lines and have shown positive correlations with 2D-DIGE data. In summary, this study identifies numerous putative breast cancer markers from various stages of breast cancer. The results of this study may aid in developing proteins identified as useful diagnostic and therapeutic candidates in research on cancer and proteomics.

Erlotinib (Tarceva) is a selective epidermal growth factor receptor tyrosine kinase inhibitor in the treatment of human non–small cell lung cancer (NSCLC). In this study, we investigated the roles of ERK1/2 and AKT signaling pathways in regulating Rad51 expression and cytotoxic effects in different NSCLC cell lines treated with erlotinib. Erlotinib decreased cellular levels of phosphorylated ERK1/2, phosphorylated AKT, Rad51 protein, and mRNA in erlotinib-sensitive H1650, A549, and H1869 cells, leading to cell death via apoptosis, but these results were not seen in erlotinib-resistant H520 and H1703 cells. Erlotinib decreased Rad51 protein levels by enhancing Rad51 mRNA and protein instability. Enforced expression of constitutively active MKK1 or AKT vectors could restore Rad51 protein levels, which were inhibited by erlotinib, and decrease erlotinib-induced cytotoxicity. Knocking down endogenous Rad51 expression by si-Rad51 RNA transfection significantly enhanced erlotinib-induced cytotoxicity. In contrast, overexpression of Rad51 by transfection with Rad51 vector could protect the cells from cytotoxic effects induced by erlotinib. Blocking the activations of ERK1/2 and AKT by MKK1/2 inhibitor (U0126) and phosphoinositide 3-kinase inhibitor (wortmannin) suppressed the expression of Rad51 and enhanced the erlotinib-induced cell death in erlotinib-resistant cells. In conclusion, suppression of Rad51 may be a novel therapeutic modality in overcoming drug resistance of erlotinib in NSCLC. (Mol Cancer Res 2009;7(8):1378–89)

Gefitinib (Iressa, ZD1839) is a selective epidermal growth factor receptor tyrosine kinase inhibitor that can block growth factor-mediated cell proliferation and extracellular signal-regulated kinases 1/2 (ERK1/2) activation. High-level Rad51 expression has been reported in chemoresistant or radioresistant carcinomas. In this study, we examined the role of Rad51 in regulating the response to gefitinib among different human lung cancer cell lines. The H520 line (human squamous cell carcinoma) was less sensitive to gefitinib compared with the H1650 (human adenocarcinoma) or A549 (human bronchioloalveolar carcinoma) lines. In H1650 and A549 cells but not in H520 cells, gefitinib decreased cellular levels of phospho-ERK1/2 and Rad51 protein and message levels. Moreover, gefitinib decreased Rad51 protein levels by enhancing Rad51 protein instability through 26S proteasome-mediated degradation. Inhibition of endogenous Rad51 levels by si-Rad51 RNA transfection significantly enhanced gefitinib-induced cytotoxicity. In contrast, transfection with constitutively active MKK1 vector could restore both Rad51 protein levels and cell survival inhibited by gefitinib. The MKK1/2-ERK1/2 signaling pathway constitutes the upstream signaling for maintaining Rad51 message and protein levels. Rad51 protein can protect lung cancer cells from cytotoxic effects induced by gefitinib. Suppression of Rad51 may be a novel lung cancer therapeutic modality to overcome drug resistance to gefitinib. [Mol Cancer Ther 2008;7(11):3632–41]

Research has shown drug resistance as the major cause of failure of cancer chemotherapy. In this study, doxorubicin-sensitive human uterine cancer cell (hUCC) MES/SA, as well as doxorubicin-resistant hUCC MES/SA-DxR 2μM and MES/SA-DxR 8μM were used. Subsequently, asparagine synthetase (ASNS), a protein that had previously been proposed to be a putative cancer drug target in our laboratory, was silenced by siRNA knockdown to study the mechanism of doxorubicin-induced resistance further. After potent knockdown of ASNS, cell viability in two doxorubicin-resistant cell lines MES/SA-DxR 2μM and MES/SA-DxR 8μM was decreased, as indicated by an MTT cell proliferation assay. By coupling two-dimensional differential gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry, proteins that play a vital role in ASNS signaling network and development of doxorubicin-induced resistance were identified. Among all the proteins that we have identified, GRP78 and AKR1C1 appear to be involved in drug resistance, replication factor C appears to participate in DNA repairing, and PP6C is proposed to play a role in cell cycle arrest.

Rhein is a natural product purified from herbal plants such as Rheum palmatum, which has been shown to have anti-angiogenesis and anti-tumor metastasis properties. However, the biological effects of rhein on the behavior of breast cancers are not completely elucidated. To evaluate whether rhein might be useful in the treatment of breast cancer and its cytotoxic mechanism, we analyzed the impact of rhein treatment on differential protein expression as well as redox regulation in a non-invasive breast cancer cell line, MCF-7, and an invasive breast cancer cell line, MDA-MB-231, using lysine- and cysteine-labeling two-dimensional difference gel electrophoresis (2D-DIGE) combined with MALDI-TOF/TOF mass spectrometry. This proteomic study revealed that 73 proteins were significantly changed in protein expression; while 9 proteins were significantly altered in thiol reactivity in both MCF-7 and MDA-MB-231 cells. The results also demonstrated that rhein-induced cytotoxicity in breast cancer cells mostly involves dysregulation of cytoskeleton regulation, protein folding, the glycolysis pathway and transcription control. A further study also indicated that rhein promotes misfolding of cellular proteins as well as unbalancing of the cellular redox status leading to ER-stress. Our work shows that the current proteomic strategy offers a high-through-put platform to study the molecular mechanisms of rhein-induced cytotoxicity in breast cancer cells. The identified differentially expressed proteins might be further evaluated as potential targets in breast cancer therapy.

Chemotherapy is one of the major categories of medical oncology and a primary tumor treatment; however, the effectiveness of chemotherapy is restricted by drug resistance. Overcoming resistance to chemotherapy and investigating molecular targeted therapies are challenges currently faced during resistance management. Progesterone receptor membrane component 1 (PGRMC1) is an adapter protein mediating cholesterol synthesis, steroid signaling, and cytochrome p450 activation. Attention has recently focused on the role of PGRMC1 in cell survival, anti-apoptosis, and damage response. In the present study, we used knockdown and overexpression approaches in the following set of uterine sarcoma models to further evaluate the role of PGRMC1 in drug resistance: the doxorubicin-sensitive MES-SA cells and the doxorubicin-resistant MES-SA/DxR-2 µM and MES-SA/DxR-8 µM cells (with different levels of doxorubicin resistance). PGRMC1 repressed doxorubicin-induced cytotoxicity and exhibited an anti-apoptotic effect; it also promoted cell proliferation and cell cycle progression to the S phase. Of note, PGRMC1 overexpression led to the epithelial-mesenchymal transition (EMT) of the sensitive MES-SA cells, thus facilitating their migration and invasion. The combination of PGRMC1 knockdown and the P-glycoprotein inhibitor verapamil significantly decreased the viability of P-glycoprotein-overexpressing MES-SA/DxR-8 μM cells after doxorubicin treatment. Taken together, our results show that PGRMC1 contributed to chemoresistance through cell proliferation, anti-apoptosis, and EMT induction, leading to the suggestion that PGRMC1 may serve as a therapeutic target in combination with an inhibitor in different drug resistance pathways and indicating the usefulness of predictive resistance biomarkers in uterine sarcoma.

The creep properties of AS-4/PEEK [ ± 45]4s laminated composites have been investigated. The temperature dependence of the viscoelastic behavior of the matrix composite and the bulk resin was studied by dynamic mechanical analysis. The tensile properties were measured at elevated temperature. The tensile failure morphology at elevated temperature was investigated by scanning electron microscopy. The accelerated characterization of the creep response due to the effect of temperature and stress was studied. Short-term creep tests were performed at various stress levels and at elevated temperatures. The creep strain data were fitted by using the Findley equation. It was found that results predicted by the Findley equation showed good agreement with the experimental data. Coupling with the Findley equation, a master curve was constructed by using the time-temperature-stress superposition principle (TTSSP) with a reference condition at 85°C and 94.38 MPa. From the short-term creep test obtained after 600 min, a smooth master curve was obtained by the proposed procedure of accelerated characterization and a creep strain of 106.29 min can be predicted.

Doxorubicin is an anticancer drug used in a wide range of cancer therapies, yet some side effects have been reported. One of these is cardiotoxicity, including cardiomyopathy and ultimately congestive heart failure. This damage to the heart has been shown to result from doxorubicin-induced reactive oxygen species. However, the cellular targets of doxorubicin-induced oxidative damage on cardiomyocytes are largely unknown. For this, a cysteine-labeling-based two-dimensional difference gel electrophoresis (2D-DIGE) combined with MALDI-TOF/TOF mass spectrometry (MALDI-TOF/TOF MS) were employed to analyze the impact of doxorubicin treatment on the redox regulation in rat cardiomyocytes. This study demonstrated 25 unique protein features that had significantly changed in their thiol reactivity and revealed that doxorubicin-induced cardiotoxicity involves dysregulation of protein folding, translational regulation and cytoskeleton regulation. Our work shows that this combined proteomic strategy provides a rapid method to study the molecular mechanisms of doxorubicin-induced cytotoxicity in the heart. The identified targets may be useful for further evaluation as potential cardiotoxic biomarkers during damage to the heart induced by doxorubicin, as well as possible diagnostic or therapeutic applications.

Quercetin, a polyphenolic compound existing in many vegetables, fruits, has antiinflammatory, antiproliferation, and antioxidant effect on mammalian cells. Quercetin was evaluated for protecting cardiomyocytes from ischemia/reperfusion injury, but its protective mechanism remains unclear in the current study. The cardioprotective effects of quercetin are achieved by reducing the activity of Src kinase, signal transducer and activator of transcription 3 (STAT3), caspase 9, Bax, intracellular reactive oxygen species production, and inflammatory factor and inducible MnSOD expression. Fluorescence two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) can reveal the differentially expressed proteins of H9C2 cells treated with H2O2or quercetin. Although 17 identified proteins were altered in H2O2-induced cells, these proteins such as alpha-soluble NSF attachment protein (α-SNAP), Ena/VASP-like protein (Evl), and isopentenyl-diphosphate delta-isomerase 1 (Idi-1) were reverted by pretreatment with quercetin, which correlates with kinase activation, DNA repair, lipid, and protein metabolism. Quercetin dephosphorylates Src kinase in H2O2-induced H9C2 cells and likely blocks the H2O2-induced inflammatory response through STAT3 kinase modulation. This probably contributes to prevent ischemia/reperfusion injury in cardiomyocytes.

Diabetic retinopathy can cause poor vision and blindness. Previous research has shown that high blood glucose weakens retinal capillaries and induces glycoxidation. However, the detailed molecular mechanisms underlying the effects of high blood glucose on development of diabetic retinopathy have yet to be elucidated. In this study, we cultured a retinal pigmented epithelium cell line (ARPE-19) in mannitol-balanced 5.5 mM, 25 mM, and 100 mM d-glucose media, and evaluated protein expression and redox-regulation. We identified 56 proteins that showed significant changes in protein expression, and 33 proteins showing significant changes in thiol reactivity, in response to high glucose concentration. Several proteins that are involved in signal transduction, gene regulation, and transport showed significant changes in expression, whereas proteins involved in metabolism, transport, and cell survival displayed changes in thiol reactivity. Further analyses of clinical plasma specimens confirmed that the proteins lamin B2, PUMA, WTAP, ASGR1, and prohibitin 2 showed type 2 diabetic retinopathy-dependent alterations. In summary, in this study, we used a comprehensive retinal cell-based proteomic approach for the identification of changes in protein expression and redox-associated retinal markers induced by high glucose concentration. Some of the identified proteins have been validated with clinical samples and provide potential targets for the prognosis and diagnosis of diabetic retinopathy.

Within ultraviolet radiation, ultraviolet B (UVB) is the most energetic and damaging to humans. At the protein level, UVB irradiation downregulates the expression of antioxidant enzymes leading to the accumulation of reactive oxygen species (ROS). Due to lacking of a global analysis of UVB-modulated corneal proteome, we investigate in vitro the mechanism of UVB-induced corneal damage to determine whether hyaluronic acid (HA) is able to reduce UVB irradiation-induced injury in human corneal epithelial cells. Accordingly, human corneal epithelial cell lines (HCE-2) were irradiated with UVB, followed by incubation with low molecular weight HA (LMW-HA, 100 kDa) or high molecular weight HA (HMW-HA, 1,000 kDa) to investigate the physiologic protection of HMW-HA in UVB-induced corneal injury, and to perform a global proteomic analysis. The data demonstrated that HA treatment protects corneal epithelial cells in the UVB-induced wound model, and that the molecular weight of HA is a crucial factor. Only HMW-HA significantly reduces the UVB-induced cytotoxic effects in corneal cells and increases cell migration and wound-healing ability. In addition, proteomic analysis showed that HMW-HA might modulate cytoskeleton regulation, signal transduction, biosynthesis, redox regulation, and protein folding to stimulate wound healing and to prevent these UVB-damaged cells from cell death. Further studies evidenced membrane-associated progesterone receptor component 1 (mPR) and malate dehydrogenase (MDH2) play essential roles in protecting corneal cells from UVB irradiation. This study reports on UVB-modulated cellular proteins that might play an important role in UVB-induced corneal cell injury and show HMW-HA to be a potential substance for protecting corneal cells from UVB-induced injury.

Oral squamous cell carcinoma (OSCC) is an aggressive cancer and its occurrence is closely related to betel nut chewing in Taiwan. However, there are few prognostic and diagnostic biomarkers for this disease especially for its association with betel nut chewing. Recent progresses in quantitative proteomics have offered opportunities to discover plasma proteins as biomarkers for tracking the progression and for understanding the molecular mechanisms of OSCC. In present study, plasma samples from OSCC patients with at least 5-year history of betel nut chewing and healthy donors were analyzed by fluorescence 2D-DIGE-based proteomic analysis. Totally, 38 proteins have been firmly identified representing 13 unique gene products. These proteins mainly function in inflammatory responses (such as fibrinogen gamma chain) and transport (Apolipoprotein A-I). Additionally, the current quantitative proteomic approach has identified numerous OSCC biomarkers including fibrinogen (alpha/beta/gamma) chain, haptoglobin, leucine-rich alpha-2-glycoprotein and ribosomal protein S6 kinase alpha-3 (RSK2) which have not been reported and may be associated with the progression and development of the disease. In summary, this study reports a comprehensive patient-based proteomic approach for the identification of potential plasma biomarkers in OSCC. The potential of utilizing these markers for screening and treating OSCC warrants further investigations.

Drug resistance is a common cause of failure in cancer chemotherapy treatments. In this study, we used a pair of uterine sarcoma cancer lines, MES-SA, and the doxorubicin-resistant MES-SA/Dx5 as a model system to examine resistance-dependent cellular responses and to identify potential therapeutic targets. We used two-dimensional differential gel electrophoresis (2D-DIGE) and matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI-TOF/TOF MS) to examine the global protein expression changes induced by doxorubicin treatment and doxorubicin resistance. A proteomic study revealed that doxorubicin-exposure altered the expression of 87 proteins in MES-SA cells, while no significant response occurred in similarly treated MES-SA/Dx5 cells, associating these proteins with drug specific resistance. By contrast, 37 proteins showed differential expression between MES-SA and MES-SA/Dx5, indicating baseline resistance. Further studies have used RNA interference, cell viability analysis, and analysis of apoptosis against asparagine synthetase (ASNS) and membrane-associated progesterone receptor component 1 (mPR) proteins, to monitor and evaluate their potency on the formation of doxorubicin resistance. The proteomic approach allowed us to identify numerous proteins, including ASNS and mPR, involved in various drug-resistance-forming mechanisms. Our results provide useful diagnostic markers and therapeutic candidates for the treatment of doxorubicin-resistant uterine cancer.

Retinopathy has been observed in around quarter of diabetic patients. Diabetic retinopathy can result in poor vision and even blindness since high glucose has been evidenced to weaken retinal capillary leading to leakage of blood into the surrounding space. In the present study, a proteomics-based approach has been applied to analyze a model retinal pigmented epithelium cell line, ARPE-19, grown in mannitol-balanced 5.5mM, 25 mM and 100 mM D-glucose culture media and used as a model for hyperglycemia secretomic analysis. Totally, 55 differentially secreted proteins have been firmly identified representing 46 unique gene products. These secreted proteins mainly function in cytoskeleton-associated adhesion/junction (such as galectin-3-binding protein) and transport (multidrug resistance-associated protein 1). Additionally, the identified secreted markers including asialoglycoprotein receptor 1, lysophosphatidic acid receptor 3, moesin, MPP2, haptoglobin and cathepsin D were further validated in plasma samples coming from type 2 diabetic patients with retinopathy and healthy donors. In summary, we report a comprehensive retinal cell-based proteomic approach for the identification of potential secreted retinal markers-induced in high glucose conditions. Some of these identified secreted proteins have been validated in diabetic retinopathy plasma demonstrating the potentially utilizing of these markers in screening and treating diabetic retinopathy.

Type 1 diabetes mellitus (T1DM) is an insulin-dependent metabolic disease in the world and often occurs in children and adolescents. Recent advances in quantitative proteomics offer potential for the discovery of plasma proteins as biomarkers for tracking disease progression and for understanding the molecular mechanisms of diabetes. Comparative proteomic analysis of the plasma proteomes from T1DM cases and healthy donors with lysine- and cysteine-labeling 2D-DIGE combining MALDI–TOF/TOF mass spectrometry revealed that 39 identified T1DM-associated plasma proteins showed significant changes in protein expression including hemopexin, and 41 in thiol reactivity. Further study showed that hemopexin can be induced in numerous cell lines by increasing the glucose concentration in the medium. Interestingly, glucose-induced hemopexin expression can be reduced by reactive oxygen species (ROS) scavengers such as glutathione, implying that hemopexin expression is linked to glucose-induced oxidative stress. In conclusion, the current work has identified potential T1DM biomarkers and one of these, hemopexin, can be modulated by glucose through a ROS-dependent mechanism.

Recent progresses in quantitative proteomics have offered opportunities to discover plasma proteins as biomarkers for tracking the progression and for understanding the molecular mechanisms of uterine leiomyomas. In the present study, plasma samples were analyzed by fluorescence two-dimensional differential gel electrophoresis (2D-DIGE) and differentially expressed proteins were identified by matrix assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). In total, 20 proteins have been firmly identified representing 13 unique gene products. These proteins mainly functioned in transportation (such as apolipoprotein A-I) and coagulation (such as fibrinogen gamma chain). Additionally, our quantitative proteomic approach has identified numerous previous reported plasma markers of uterine leiomyomas such as alpha-1-antitrypsin. On the contrary, we have presented several putative uterine leiomyomas biomarkers including afamin, apolipoprotein A-I, carbonic anhydrase 1, fibrinogen beta chain, fibrinogen gamma chain, gelsolin, hemopexin, leucine-rich alpha-2-glycoprotein, serotransferrin and vitamin D-binding protein which have not been reported and may be associated with the progression and development of the disease. In summary, we report a comprehensive patient-based proteomic approach for the identification of potential plasma biomarkers for uterine leiomyomas. The potential of utilizing these markers for screening and treating uterine leiomyomas warrants further investigations.

UVB is the most energetic and DNA-damaging to humans in ultraviolet radiation. Previous research has suggested that exposure to UVB causes skin pathologies because of direct DNA damage and the generation of reactive oxygen species (ROS). However, the detailed molecular mechanisms by which UVB leads to skin cancer have yet to be clarified. In the current study, normal skin fibroblast cells (CCD-966SK) were exposed to various doses of UVB, and the changes in protein expression and thiol reactivity were monitored with lysine- and cysteine-labeling 2D-DIGE and MALDI-TOF mass spectrometry. Our proteomic analysis revealed that 89 identified proteins showed significant changes in protein expression, and 37 in thiol reactivity. Many proteins that are known to be involved in protein folding, redox regulation and nucleotide biosynthesis were up-regulated under UVB irradiation. In contrast, proteins responsible for biosynthesis and protein degradation were down-regulated. In addition, the thiol-reactivity of proteins involving cytoskeleton, metabolism, and signal transduction were altered by UVB. In summary, these UVB-modulated cellular proteins and redox-regulated proteins might play important roles in the early stages of skin cancer formation and photoaging induced by UVB-irradiation. Such proteins might provide a potential target for the rational design of drugs to prevent UVB-induced diseases.

Emodin (1,3,8-trihydroxy-6-methyl-anthraquinone) is a natural anthraquinone derivative found in the roots and rhizomes of numerous plants. It is a tyrosine kinase inhibitor and has anticancer effects on lung cancer. Rad51 plays a central role in homologous recombination, and high levels of Rad51 expression are observed in chemo- or radioresistant carcinomas. Our previous studies have shown that the mitogen-activated protein kinase kinase (MKK) 1/2-extracellular signal-regulated kinase (ERK) 1/2 signal pathway maintains the expression of Rad51. Therefore, in this study, we hypothesized that emodin could enhance the effects of the antitumor antibiotic mitomycin C (MMC)-mediated cytotoxicity by decreasing the expression of Rad51 and the phosphorylation of ERK1/2. Exposure of the human non-small-cell lung cancer H1703 or A549 cell lines to emodin decreased the MMC-elicited phosphorylated ERK1/2 and Rad51 levels. Moreover, emodin significantly decreased the MMC-elicited Rad51 mRNA and protein levels by increasing the instability of Rad51 mRNA and protein. In emodin- and MMC-cotreated cells, ERK1/2 phosphorylation was enhanced by constitutively active MKK1/2 (MKK1/2-CA), thus increasing Rad51 protein levels and protein stability. The synergistic cytotoxic effects induced by emodin combined with MMC were remarkably decreased by MKK1-CA-mediated enhancement of ERK1/2 activation. Depletion of endogenous Rad51 expression by small interfering Rad51 RNA transfection significantly enhanced MMC-induced cell death and cell growth inhibition. In contrast, overexpression of Rad51 protects lung cancer cells from the synergistic cytotoxic effects induced by emodin and MMC. We conclude that suppression of Rad51 expression or a combination of emodin with chemotherapeutic agents may be considered as potential therapeutic modalities for lung cancer.

Emodin, a tyrosine kinase inhibitor, is a natural anthraquinone derivative found in the roots and rhizomes of numerous plants. It reportedly exhibits an anticancer effect on lung cancer. Gefitinib (Iressa) is a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor for human non-small cell lung cancer (NSCLC). However, the molecular mechanism of how emodin combined with gefitinib decreases NSCLC cell viability is unclear. The recombinase protein Rad51 is essential for homologous recombination repair, and Rad51 overexpression is resistant to DNA double-strand break-inducing cancer therapies. In this study, we found that emodin enhanced the cytotoxicity induced by gefitinib in two NSCLC cells lines, A549 and H1650. Emodin at low doses of 2-10 microM did not affect ERK1/2 activation, mRNA, and Rad51 protein levels; however, it enhanced a gefitinib-induced decrease in phospho-ERK1/2 and Rad51 protein levels by enhancing Rad51 protein instability. Expression of constitutively active MKK1/2 vectors (MKK1/2-CA) significantly rescued the reduced phospho-ERK1/2 and Rad51 protein levels as well as cell viability on gefitinib and emodin cotreatment. Blocking of ERK1/2 activation by U0126 (an MKK1/2 inhibitor) lowered Rad51 protein levels and cell viability in emodin-treated H1650 and A549 cells. Knockdown of Rad51 expression by transfection with si-Rad51 RNA enhanced emodin cytotoxicity. In contrast, Rad51 overexpression protected the cells from the cytotoxic effects induced by emodin and gefitinib. Consequently, emodin-gefitinib cotreatment may serve as the basis for a novel and better therapeutic modality in the management of advanced lung cancer.

Celecoxib (Celebrex R ) is a cyclooxygenase-2 (COX-2) selective inhibitor and gefitinib (Iressa R , ZD1839) is a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor for human non-small cell lung cancer (NSCLC). The addition of celecoxib to gefitinib to prolong the survival of patients with NSCLC still remains controversial and needs to be investigated. The Rad51 protein is essential for homologous recombination repair, and is overexpressed in chemo- or radioresistant carcinomas. In this study, we characterize the role of celecoxib in the cytotoxicity, ERK1/2 activation and Rad51 expression affected by gefitinib in NSCLC cells. We show that celecoxib can enhance the cytotoxicity induced by gefitinib in NSCLC cells. Treatment with celecoxib alone has no effect on the ERK1/2 activation, Rad51 mRNA and protein levels, however, combined treatment with gefitinib results in a significant reduction of phospho-ERK1/2 and Rad51 protein levels, and triggers the degradation of Rad51 via a 26S proteasome-dependent pathway. Expression of constitutively active MKK1/2 vectors (MKK1/2-CA) significantly rescues the decreased ERK1/2 activity, and restores Rad51 protein levels and cell survival under co-treatment with gefitinib and celecoxib. Furthermore, blocking ERK1/2 activation by U0126 (MKK1/2 inhibitor) and knocking down Rad51 expression by transfection with small interfering RNA of Rad51 can enhance the cytotoxicity of celecoxib.

Static tensile strength and fatigue behavior of long glass fiber reinforced semicrystalline polyamide(Nylon6,6) and amorphous polycarbonate(PC) composites were investigated. The static tensile measurement at various temperatures and tension-tension fatigue loading tests at various levels of stress amplitudes were studied. Two-parameters Weibull distribution function and pooled Weibull distribution function were applied to obtain the statistical probability distribution of experimental data of static tensile strength and fatigue life under different stress amplitude tests. The dynamic creep property and the temperature increasing phenomenon under tension-tension fatigue loading were compared between semicrystalline and amorphous composites. Results showed that the static tensile strength of polyamide composites is higher than that of polycarbonate composites, with lower fatigue life and more sensitive to temperature. The slope of S-N0 curves of long glass fiber reinforced semicrystalline polyamide and amorphous polycarbonate composites were almost identical.

Currently, the most effective agent against pancreatic cancer is gemcitabine (GEM), which inhibits tumor growth by interfering with DNA replication and blocking DNA synthesis. However, GEM-induced drug resistance in pancreatic cancer compromises the therapeutic efficacy of GEM. To investigate the molecular mechanisms associated with GEM-induced resistance, 2D-DIGE and MALDI-TOF mass spectrometry were performed to compare the proteomic alterations of a panel of differential GEM-resistant PANC-1 cells with GEM-sensitive pancreatic cells. The proteomic results demonstrated that 33 proteins were differentially expressed between GEM-sensitive and GEM-resistant pancreatic cells. Of these, 22 proteins were shown to be resistance-specific and dose-dependent in the regulation of GEM. Proteomic analysis also revealed that proteins involved in biosynthesis and detoxification are significantly over-expressed in GEM-resistant PANC-1 cells. In contrast, proteins involved in vascular transport, bimolecular decomposition, and calcium-dependent signal regulation are significantly over-expressed in GEM-sensitive PANC-1 cells. Notably, both protein-protein interaction of the identified proteins with bioinformatic analysis and immunoblotting results showed that the GEM-induced pancreatic cell resistance might interplay with tumor suppressor protein p53. Our approach has been shown here to be useful for confidently detecting pancreatic proteins with differential resistance to GEM. Such proteins may be functionally involved in the mechanism of chemotherapy-induced resistance.