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PM2.5, generated via both direct emission and secondary formation, can have varying environmental impacts due to different physical and chemical properties of its components. However, traditional methods to quantify different PM2.5 components are often based on online or offline observations and numerical models, which are generally high economic cost- or labor-intensive. In this study, we develop a new method, named Multi-Tracer Estimation Algorithm (MTEA), to identify the primary and secondary components from routine observation of PM2.5. By comparing with long-term and short-term measurements of aerosol chemical components in China and the United States, it is proven that MTEA can successfully capture the magnitude and variation of the primary PM2.5 (PPM) and secondary PM2.5 (SPM). Applying MTEA to the China National Air Quality Network, we find that (1) SPM accounted for 63.5 % of the PM2.5 in cities in southern China on average during 2014–2018, while the proportion dropped to 57.1 % in the north of China, and at the same time the secondary proportion in regional background regions was ∼ 19 % higher than that in populous regions; (2) the summertime secondary PM2.5 proportion presented a slight but consistent increasing trend (from 58.5 % to 59.2 %) in most populous cities, mainly because of the recent increase in O3 pollution in China; (3) the secondary PM2.5 proportion in Beijing significantly increased by 34 % during the COVID-19 lockdown, which might be the main reason for the observed unexpected PM pollution in this special period; and finally, (4) SPM and O3 showed similar positive correlations in the Beijing-Tianjin-Hebei (BTH) and Yangtze River Delta (YRD) regions, but the correlations between total PM2.5 and O3 in these two regions, as determined from PPM levels, were quite different. In general, MTEA is a promising tool for efficiently estimating PPM and SPM, and has huge potential for future PM mitigation.

The Clean Air Plan has been active in China since 2013 to mitigate severe PM

A sensitive and selective Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method was developed for the identification and quantification of two potential genotoxic impurities (PGIs) - viz. methyl N-((2'-(1H-tetrazol-5-yl)-[1,1'-biphenyl]-4-yl)methyl)-N-nitroso-L-valinate (PGI-1) and N-nitroso Valsartan (PGI-2) - in the angiotensin II receptor blocker valsartan. Among these impurities, PGI-1 is a distinctive compound which has never been reported. For this, chromatographic separation was performed using a Waters XBridge BEH C18 column (150 mm × 4.6 mm, 2.5 µm), with ammonium acetate aqueous solution (0.01 mol/L) as mobile phase A and acetonitrile as mobile phase B, in a gradient elution mode at a 0.5 mL/min flow rate. Mass spectrometric conditions were optimized using electrospray ionization (ESI) in positive mode. Following the International Conference of Harmonization (ICH) guidelines, this methodology is capable of quantifying 2 PGIs at 0.016 ppm in samples at 50 mg/mL concentration. This validated approach presented good linearity over the concentration range of 0.016-0.06 ppm for 2 PGIs. The correlation coefficient of each impurity was observed greater than 0.999. The accuracy of this method was in the range of 83-113% for the aforementioned PGIs. In addition, expert knowledge rules (Derek-based) and statistical (Q) SAR evaluation system (Sarah-based) were used to evaluate and classify the genotoxicity of both valsartan-related PGIs as well as to define their standard limits. The predicted results were positive and classified into the third category, and the total nitrosamine limit was set to 0.03 ppm. As such, this approach represents a good quality control system for the simultaneous and precise quantitation of PGIs in valsartan.

PM2.5, generated via both direct emissions and secondary formations, can have varying environmental impacts due to different physical and chemical properties of its components. However, traditional methods to quantify different PM2.5 components are often based on online observations or lab analyses, which are generally high economic cost and labor-intensive. In this study, we develop a new method, named multi-tracer estimation algorithm (MTEA), to identify the primary and secondary components from routine observation of PM2.5. By comparing with the long-term and short-term measurements of aerosol chemical components in China, as well as aerosol composition network in the United States, MTEA is proved to be able to successfully capture the magnitude and variation of the primary PM2.5 (PPM) and secondary PM2.5 (SPM). Applying MTEA to China national air quality network, we find that 1) SPM accounts for 63.5 % of PM2.5 in southern cities of China averaged for 2014–2018, while in the North the proportion drops to 57.1 %, and at the same time the secondary proportion in regional background regions is ~19 % higher than that in populous regions; 2) the summertime secondary PM2.5 proportion presents a slight but consistent increasing trend (from 58.5 % to 59.2 %) in most populous cities, mainly because of the recent increase in O3 pollution in China; 3) the secondary PM2.5 proportion in Beijing significantly increases by 34 % during the COVID-19 lockdown, which might be the main reason of the observed unexpected PM pollution in this special period; and at least, 4) SPM and O3 show similar positive correlations in the BTH and YRD regions, but the correlations between total PM2.5 and O3 in these two regions are quite different as PPM levels determines. In general, MTEA is a promising tool for efficiently estimating PPM and SPM, and has huge potential for the future PM mitigation.

Xylaneses are very common xylanolytic enzymes, which are widely used in food, papermaking, and other industries. In this study, a xylanase-encoding gene xyn1923, which encodes a protein of 1352 amino acids, was identified through the whole genome analysis of Microbacterium imperiale YD-01. Bioinformatics analysis showed that Xyn1923 only had maximum similarity of 37% with the reported xylanase from Alkalihalobacillus halodurans C-125, indicating that Xyn1923 was a novel xylanase. The enzymatic properties of Xyn1923 were systematically analyzed after purification. The results showed that the specific activity of the enzyme was 10.582 ± 0.413 U/mg, while the optimum pH and temperature of the enzyme were 7.0 and 70°C, respectively. The enzyme is stable in the pH range of 6.0-9.0, and the enzyme activity could maintain more than 85% of the original activity after 16 hr incubation at pH 9.0. The enzyme activity is relatively stable in the range of 30-60°C, and its enzyme activity could maintain more than 89% of the original activity after treatment at 60°C for 30 min. Low concentrations (≤1 mM) of Co2+ , Ba2+ , Fe2+ , and Fe3+ metal ions exerted a stimulatory effect on the activity of Xyn1923. And in contrast, high concentrations (≥2 mM) of the above metal ions inhibit the activity of Xyn1923. Mg2+ , Ag+ , Cu2+ , Ca2+ , Mn2+ , and Pb2+ ions showed a negative effect on the activity of Xyn1923. Enzyme kinetic studies showed that Km and Vmax values for xylan were 7.842 ± 0.538 mg/ml and 15.208 ± 0.822 U/mg, respectively. Xyn1923 was found to be a weakly alkaline thermophilic xylanase through an enzymatic property analysis. PRACTICAL APPLICATIONS: Xylanases are widely used in food and feed, biofuels, papermaking, and other industries. However, their use is limited by poor performance under the conditions of pH and temperature. Therefore, the discovery of xylanases with the capability of working efficiently at alkaline pH and high temperature is the priority for its industrial applications. In this study, a novel xylanase-encoding gene xyn1923 from Microbacterium imperiale YD-01 was cloned and heterologously expressed in Escherichia coli. Enzymatic properties of this novel xylanase were investigated, indicating that the robust thermal stability and alkali resistance of Xyn1923 make it a potential candidate for the food and paper industries.

Satellite-based inverse modeling has the potential to drive aerosol precursor emissions, but its efficacy for improving chemistry transport models (CTMs) remains elusive because of its likely inherent dependence on the error characteristics of a specific CTM used for the inversion. This issue is quantitively assessed here by using three CTMs. We show that SO2 emissions from global GEOS-Chem adjoint model and OMI SO2 data, when combined with spatial variation of bottom-up emissions, can largely improve WRF-Chem and WRF-CMAQ forecast of SO2 and aerosol optical depth (in reference to moderate resolution imaging spectroradiometer data) in China. This suggests that the efficacy of satellite-based inversion of SO2 emission appears to be high for CTMs that use similar or identical emission inventories. With the advent of geostationary air quality monitoring satellites in next 3 years, this study argues that an era of using top-down approach to rapidly update emission is emerging for regional air quality forecast, especially over Asia having highly varying emissions.

12-Lipoxygenase (12-LOX) metabolizes arachidonic acid to 12(S)-hydroxyeicosatetraenoic acid, or 12(S)-HETE, a proinflammatory bioactive lipid implicated in tumor angiogenesis, growth, and metastasis. The mechanisms underlying 12-LOX-mediated signaling in cancer progression are still ill-defined. In the present study we demonstrate that 12-LOX phosphorylation and subsequent enzymatic activity occurs after integrin β4 stimulation and Src kinase recruitment to the integrin subunit. Inhibition of Src activity by PP2 or Src dominant-negative mutants reduced 12-LOX tyrosine phosphorylation and 12(S)-HETE production in response to integrin β4 stimulation in A431 cells. The pertinent Src-targeted residues for 12-LOX activity were mapped to Y19 and Y614, where 12-LOX mutants Y19F and Y614F showed 70% less enzymatic activity. Furthermore, we have shown that the 12-LOX activity modulated by these residues impacts migration. To our knowledge, this is the first report that c-Src kinase activity is required for β4-integrin-mediated phosphorylation of 12-LOX.

International audience; 12-Lipoxygenase utilizes arachidonic acid to synthesize 12(S)-hydroperoxyeicosatetraenoic acid, which is converted to the end product 12(S)-hydroxyeicosatetraenoic acid, an eicosanoid that promotes tumorigenesis and metastasis. Increased expression of 12-lipoxygenase has been documented in a number of carcinomas. When overexpressed in human prostate or breast cancer, 12-lipoxygenase promotes tumor angiogenesis and growth in vivo. The present study was undertaken to delineate the mechanisms by which 12-lipoxygenase enhances angiogenesis. Herein we report that nordihydroguaiaretic acid, a pan inhibitor of lipoxygenases and baicalein, a selective inhibitor of 12-lipoxygenase, reduced VEGF expression in human prostate cancer PC-3 cells. Overexpression of 12-lipoxygenase in PC-3 cells resulted in a 3-fold increase in VEGF protein level when compared with vector control cells. An increase in PI 3-kinase activity was found in 12-LOX-transfected PC-3 cells and inhibition of PI 3-kinase by LY294002 significantly reduced VEGF expression. Northern blot and real time PCR analyses revealed an elevated VEGF transcript level in PC-3 cells transfected with a 12-lipoxygenase expression construct. Using a VEGF promoter luciferase construct (-1176/+54), we found a 10-fold increase in VEGF promoter activity in 12-lipoxygenase-transfected PC-3 cells. The region located between -88 and -66 of the VEGF promoter was identified as 12-lipoxygenase responsive using VEGF promoter-based luciferase assays. Further analysis with mutant constructs indicated Sp1 as a transcription factor required for 12-lipoxygenase stimulation of VEGF. Neutralization of VEGF by a function-blocking antibody significantly decreased the ability of 12-lipoxygenase-transfected PC-3 cells to stimulate endothelial cell migration, suggesting VEGF as an important effector for 12-lipoxygenase-mediated stimulation of tumor angiogenesis.

Metabolism of arachidonic acid through cyclooxygenase, lipoxygenase, or P450 epoxygenase pathways leads to the formation of various bioactive eicosanoids. In this review, we discuss alterations in expression pattern of eicosanoid-generating enzymes found during prostate tumor progression and expound upon their involvement in tumor cell proliferation, apoptosis, motility, and tumor angiogenesis. The expression of cyclooxygenase-2, 12-lipoxygenase, and 15-lipoxygenase-1 are up-regulated during prostate cancer progression. It has been demonstrated that inhibitors of cyclooxygenase-2, 5-lipoxygenase and 12-lipoxygenase cause tumor cell apoptosis, reduce tumor cell motility and invasiveness, or decrease tumor angiogenesis and growth. The eicosanoid product of 12-lipoxygenase, 12(S)-hydroeicosatetraenoic acid, is found to activate Erk1/2 kinases in LNCaP cells and PKCα in rat prostate AT2.1 tumor cells. Overexpression of 12-lipoxygenase and 15-lipoxygenase-1 in prostate cancer cells stimulate prostate tumor angiogenesis and growth, suggesting a facilitative role for 12-lipoxygenase and 15-lipoxygenase-1 in prostate tumor progression. The expression of 15-lipoxygenase-2 is found frequently to be lost during the initiation and progression of prostate tumors. 15(S)-hydroxyeicosatetraenoic acid, the product of 15-lipoxygenase-2, inhibits proliferation and causes apoptosis in human prostate cancer cells, suggesting an inhibitory role for 15-lipoxygenase-2 in prostate tumor progression. The regulation of prostate cancer progression by eicosanoids, in either positive or negative ways, provides an exciting possibility for management of this disease.

Prostaglandin endoperoxide H synthases and their arachidonate products have been implicated in modulating angiogenesis during tumor growth and chronic inflammation. Here we report the involvement of thromboxane A(2), a downstream metabolite of prostaglandin H synthase, in angiogenesis. A TXA(2) mimetic, U46619, stimulated endothelial cell migration. Angiogenic basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) increased TXA(2) synthesis in endothelial cells three- to fivefold. Inhibition of TXA(2) synthesis with furegrelate or CI reduced HUVEC migration stimulated by VEGF or bFGF. A TXA(2) receptor antagonist, SQ29,548, inhibited VEGF- or bFGF-stimulated endothelial cell migration. In vivo, CI inhibited bFGF-induced angiogenesis. Finally, development of lung metastasis in C57Bl/6J mice intravenously injected with Lewis lung carcinoma or B16a cells was significantly inhibited by thromboxane synthase inhibitors, CI or furegrelate sodium. Our data demonstrate the involvement of TXA(2) in angiogenesis and development of tumor metastasis.

To study whether alpha6beta4 integrin regulates apoptosis, human A431 cells were plated on bacteria plates in the presence or absence of mAb beta4. In the absence of mAb beta4, A431 cells demonstrated morphological characteristics of apoptosis by 24 h and most cells died by 48 h. In contrast, in the presence of mAb beta4, cells remained viable, and at the end of 48 h, 70-80% of cells survived. Treatment of A431 cells with mAb beta4 resulted in tyrosine phosphorylation of the p85 subunit of PI3 kinase; PI3 kinase activity increased within 15 min and peaked at 60 min. Stimulation of beta4 in A431 cells resulted in a time-dependent phosphorylation of Akt with a concomitant and parallel phosphorylation of Bad. Inactivation of PI3 kinase with inhibitors blocked the anti-apoptotic effect induced by mAb beta4. These are the first results to suggest that ligation of alpha6beta4 integrin protects cells from apoptosis through a PI3K/Akt kinase signaling pathway.

Arachidonic acid metabolism through cyclooxygenase, lipoxygenase, or P-450 epoxygenase pathways can generate a variety of eicosanoids. Thromboxane synthase (TxS) metabolizes the cyclooxygenase product, prostanglandin H(2), into thromboxane A(2) (TXA(2)), which can cause vessel constriction, platelet activation, and aggregation. Here we demonstrate that human prostate cancer (PCa) cells express enzymatically active TxS and that this enzyme is involved in cell motility. In human PCa cell lines, PC-3, PC-3M, and ML-2 cells expressed higher levels of TxS than normal prostate epithelial cells or other established PCa cell lines such as DU145, LNCaP, or PPC-1. We cloned and sequenced the full-length TxS cDNA from PC-3 cells and found two changes in the amino acid residues. Immunohistochemical analysis of tumor specimens revealed that expression of TxS is weak or absent in normal differentiated luminal, or secretory cells, significantly elevated in less differentiated or advanced prostate tumors, and markedly increased in tumors with perineural invasion. TxS expressed in PC-3 cells was enzymatically active and susceptible to carboxyheptal imidazole, an inhibitor of TxS. The biosynthesis of TXA(2) in PC-3 cells was dependent on COX-2, and to a lesser extent, COX-1. Treatment of PC-3 cells with a COX-1 selective inhibitor, piroxicam, reduced TXA(2) synthesis by approximately 40%, while the COX-2 specific inhibitor NS398 reduced TXA(2) production by approximately 80%. Inhibition of TxS activity or blockade of TXA(2) function reduced PC-3 cell migration on fibronectin, while having minimal effects on cell cycle progression or survival. Finally, increased expression of TxS in DU145 cells increased cell motility. Our data suggest that human PCa cells express TxS and that this enzyme may contribute to PCa progression through modulating cell motility.

Arachidonate 12-lipoxygenase (LOX) converts arachidonic acid to 12(S)-hydroxyeicosatetraenoic acid (HETE), a bioactive lipid implicated in tumor angiogenesis, growth, and metastasis. Alteration in 12-LOX expression or activity has been reported in various carcinomas including prostate carcinoma. However, little is known about the impact of the altered expression or activity of 12-LOX on tumor metastasis. In the present study, we examined whether or not an increase in 12-LOX expression in human prostate carcinoma cells can modulate their metastatic potential. We report that increased expression of 12-LOX in PC-3 cells caused a significant change in cell adhesiveness, spreading, motility, and invasiveness. Specifically 12-LOX transfected PC-3 cells were more adhesive toward vitronectin, type I and IV collagen, but not to fibronectin or laminin, than cells transfected with control vector. Increased spreading on vitronectin, fibronectin, collagen type I and IV also was observed in 12-LOX transfected PC-3 cells when compared to control PC-3 cells. The increased spreading of 12-LOX transfected PC-3 cells was blocked by treatment with 12-LOX inhibitors, baicalein and CDC. 12-LOX transfected PC-3 cells were more invasive through Matrigel than cells transfected with control vector. In vivo, tumor cell invasion to surrounding muscle or fat tissues was more frequent in nude mice bearing s.c. tumors from 12-LOX transfected PC-3 cells than in those from control vector transfected cells. When injected via the tail vein into SCID mice with implanted human bone fragments, there was an increase in tumor metastasis to human bone by 12-LOX transfected PC-3 cells in comparison to control vector transfected cells. Taken together, our data suggest that an increase in 12-LOX expression enhances the metastatic potential of human prostate cancer cells.

Arachidonic acid metabolism leads to the generation of biologically active metabolites that regulate cell growth and proliferation, as well as survival and apoptosis. We have demonstrated previously that platelet-type 12-lipoxygenase (LOX) regulates the growth and survival of a number of cancer cells. In this study, we show that overexpression of platelet-type 12-LOX in prostate cancer PC3 cells or epithelial cancer A431 cells significantly extended their survival and delayed apoptosis when cultured under serum-free conditions. These effects were shown to be a result of enhanced surface integrin expression, resulting in a more spread morphology of the cells in culture. PC3 cells transfected with 12-LOX displayed increased alpha(v)beta(3) and alpha(v)beta(5) integrin expression, whereas other integrins were unaltered. Transfected A431 cells did not express alpha(v)beta(3); however, alpha(v)beta(5) integrin expression was increased. Treatment of both transfected cell lines with monoclonal antibody to alpha(v)beta(5) (and in the case of PC3 cells, anti-alpha(v)beta(3)) resulted in significant apoptosis. In addition, treatment with 100 nM 12(S)-hydroxy-eicosatetraenoic acid, the end product of platelet-type 12-LOX, but not other hydroxy-eicosatetraenoic acids, enhanced the survival of wild-type PC3 and A431 cells and resulted in increased expression of alpha(v)beta(5). Furthermore, Baicalein or N-benzyl-N-hydroxy-5-phenylpentamide, specific 12-LOX inhibitors, significantly decreased alpha(v)beta(5)-mediated adhesion and survival in 12-LOX-overexpressing cells. The results show that 12-LOX regulates cell survival and apoptosis by affecting the expression and localization of the vitronectin receptors, alpha(v)beta(3) and alpha(v)beta(5), in two cancer cell lines.

Work functions of Cr/Ni (111), Cr/Ni (100) and Cr/Ni (110) surfaces with different magnetic configurations for Cr atoms in the topmost Cr monolayer are investigated using first-principles methods based on density functional theory. The calculated results reveal that work functions vary with crystal orientations and magnetic configurations. The magnitude of the Cr magnetic moments for the three (111), (100) and (110) surfaces follows a change trend with MCr, Cr/Ni(111)< MCr, Cr/Ni(100)< MCr, Cr/Ni(110). Altering the magnetic configurations of the systems from an original ground state to an excited one will have the total energy and the Fermi level increase. Consequently, it will give rise to the reduction of the work function for the system. Moreover, the quite favorable variation range (4.92–4.41 eV) of the calculated work functions for Cr/Ni (100) system modulated by spin effect implies that the Cr/Ni (100) system may be a more promising candidate. Our work suggests that changing magnetic configurations can modulate the work functions of magnetic metal gates well.

The magnetism and work function Φ of Fe1—xGdx/Fe (001) films have been investigated using first-principles methods based on the density functional theory. The calculated results reveal that Gd doping on the Fe (001) surface would greatly affect the geometrical structure of the system. The restruction of the surface atoms leads to the transition of magnetic coupling between Gd and Fe atoms from ferromagnetic (FM) for 0.5 ≤ x ≤ 0.75 to antiferromagnetic (AFM) for x = 1.0. For Fe1—xGdx/Fe (001) (x = 0.25, 0.5, 0.75, 1.0), the charge transfer from Gd to Fe leads to a positive dipole formed on the surface, which is responsible for the decrease of the work function. Moreover, it is found that the magnetic moments of Fe and Gd on the surface layer can be strongly influenced by Gd doping. The changes of the work function and magnetism for Fe1—xGdx/Fe (001) can be explained by the electron transfer, the magnetic coupling interaction between Gd and Fe atoms, and the complex surface restruction. Our work strongly suggests that the doping of the metal with a low work function is a promising way for modulating the work function of the magnetic metal gate.

12(S)-Hydroxyeicosatetraenoic acid (12(S)-HETE), a 12-lipoxygenase metabolite of arachidonic acid, has multiple effects on tumor and endothelial cells, including stimulation of invasion and angiogenesis. However, the signaling mechanisms controlling these physiological processes are poorly understood. In a human epidermoid carcinoma cell line (i.e. A431), 12(S)-HETE activates extracellular signal-regulated kinases 1/2 (ERK1/2), which is mediated by upstream kinases MEK and Raf. 12(S)-HETE stimulates phosphorylation of phospholipase Cgamma1 and activity of protein kinase Calpha (PKCalpha). In addition, independent of PKC 12(S)-HETE increases tyrosine phosphorylation of Shc, and Grb2, stimulates association between Shc and Src, and increases the activity of Ras, via Src family kinases. Furthermore, at low (10-100 nm) concentrations 12(S)-HETE counteracts epidermal growth factor-stimulated activation of ERK1/2 via stimulating protein tyrosine phosphatases. We also present evidence that 12(S)-HETE stimulates ERK1/2 via G proteins and that A431 cells have multiple binding sites for 12(S)-HETE. Finally, inhibition of 12-lipoxygenase induced apoptosis of A431 cells, which was reversed by addition of exogenous 12(S)-HETE. Collectively we demonstrate that the activation of ERK1/2 by 12(S)-HETE may be regulated by multiple receptors triggering PKC-dependent and PKC-independent pathways in A431 cells.

Cancer of the prostate is the most commonly diagnosed cancer in America. There are several lines of evidence implicating the involvement of arachidonate 12-lipoxygenase, an enzyme metabolizing arachidonic acid to form 12(S)-hydroxyeicosatetraenoic acid (HETE), in prostate cancer progression. First, as prostate cancer reaches a more advanced stage, the level of 12-lipoxygenase expression is increased. Second, overexpression of 12-lipoxygenase in human prostate cancer cells stimulates angiogenesis and tumor growth. Third, an inhibitor of 12-lipoxygenase has been found effective against metastatic prostate tumor growth, and the inhibition of 12-lipoxygenase is related with the reduction of tumor angiogenesis. Collectively, these studies suggest that 12-lipoxygenase regulates tumor angiogenesis in prostate cancer and that inhibition of 12-lipoxygenase is a novel therapeutic approach for the treatment of prostate cancers.

Angiogenesis, the formation of new capillaries from preexisting blood vessels, is a multistep, highly orchestrated process involving vessel sprouting, endothelial cell migration, proliferation, tube differentiation, and survival. Eicosanoids, arachidonic acid (AA)-derived metabolites, have potent biologic activities on vascular endothelial cells. Endothelial cells can synthesize various eicosanoids, including the 12-lipoxygenase (LOX) product 12(S)-hydroxyeicosatetraenoic acid (HETE). Here we demonstrate that endogenous 12-LOX is involved in endothelial cell angiogenic responses. First, the 12-LOX inhibitor, N-benzyl-N-hydroxy-5-phenylpentanamide (BHPP), reduced endothelial cell proliferation stimulated either by basic fibroblast growth factor (bFGF) or by vascular endothelial growth factor (VEGF). Second, 12-LOX inhibitors blocked VEGF-induced endothelial cell migration, and this blockage could be partially reversed by the addition of 12(S)-HETE. Third, pretreatment of an angiogenic endothelial cell line, RV-ECT, with BHPP significantly inhibited the formation of tubelike/cordlike structures within Matrigel. Fourth, overexpression of 12-LOX in the CD4 endothelial cell line significantly stimulated cell migration and tube differentiation. In agreement with the critical role of 12-LOX in endothelial cell angiogenic responses in vitro, the 12-LOX inhibitor BHPP significantly reduced bFGF-induced angiogenesis in vivo using a Matrigel implantation bioassay. These findings demonstrate that AA metabolism in endothelial cells, especially the 12-LOX pathway, plays a critical role in angiogenesis.

The platelet isoform of 12-lipoxygenase (12-LOX) is expressed in a variety of human tumors. 12-LOX metabolizes arachidonic acid to 12(S)-hydroxyeicosateraenoic acid (12(S)-HETE), which induces a number of cellular responses associated with tumor progression and metastasis. Little is known about 12-LOX regulation and no direct regulators of 12-LOX activity have been identified. To identify potential regulators of 12-LOX, we isolated cDNAs encoding 12-LOX interacting proteins using the yeast two-hybrid system. We screened a yeast two-hybrid interaction library from human epidermoid carcinoma A431 cells and identified four cellular proteins that interact specifically with 12-LOX. We identified type II keratin 5, lamin A, the cytoplasmic domain of integrin beta4 subunit and a phosphoprotein C8FW as 12-LOX interacting proteins. Here, we demonstrated that keratin 5, a 58 kD protein required for formation of 8 nm intermediate filaments, binds to 12-LOX in human tumor cells and may contribute to the regulated trafficking of 12-LOX. We also showed that lamin A binds 12-LOX in human tumor cells. These proteins provide the first candidate regulators of 12-LOX.

Metastasis is a complex process composed of sequential events involving host celltumor cell interactions, which enable tumor cells to disseminate from the primary site to distant locations (1). To successfully establish a secondary metastatic colony, tumor cells must be able to overcome all of the steps in the metastatic cascade, including detachment, intravasation, arrest, extravasation and proliferation. Ample cell-host interactions, such as tumor cell-platelet, tumor cell-endothelial cell and tumor cell-matrix protein interaction, are influenced by positive and negative regulatory factors. Eicosanoids and other bioactive lipids have been shown to be involved in various aspects of neoplasia including cell transformation, proliferation, apoptosis, invasion and metastasis. Platelets and endothelial cells with which tumor cells interaction during hematogenous metastasis are capable of producing a vast array of lipid mediators by either direct or transcellular metabolism of precursors. Platelets and some tumor cells are capable of converting arachidonic acid (AA) through the 12-lipoxygenase (12-LOX) pathway into 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE). A large collection of experimental data suggest that 12(S)-HETE plays a pivotal role in multiple steps of the metastatic cascade encompassing tumor cell-vasculature interaction, tumor cell motility, proteolysis, invasion, and angiogenesis. In this review we summarize the effects of 12(S)-HETE in modulating tumor metastasis.

The growth and metastasis of solid tumors are dependent on the ability of tumor cells to induce angiogenesis [1]. Angiogenesis, the formation of new blood vessels from pre-existing ones, involves endothelial cell proliferation, motility, and tubular differentiation. It is known that tumor cells can secrete a variety of angiogenic factors, such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), to stimulate angiogenesis. Tumor cells also produce angiogenesis inhibitors such as thrombospondin and angiostatin to control angiogenesis. The balance between angiogenesis stimulators and inhibitors determines the angiogenicity of tumor cells [2]. Acquisition of the ability to stimulate angiogenesis by tumor cells is an integral part of tumorigenesis. Activated oncogenes, such as ras, or inactivated tumor suppressor genes, such as p53, not only increase mitogenesis and prevent apoptosis in tumor cells, but also lead to the development of an angiogenic phenotype [for review,.3].

Apoptosis or programmed cell death, plays an indispensable role in embryonic development, maturation of the immune system and maintenance of tissue and organ homeostasis (1–2). A wide spectrum of molecular entities including oncogenes, tumor suppressor genes, signal transducers, cell cycle proteins, free radicals, cations and proteases have been implicated in apoptosis. Recent work from our laboratory (3) demonstrated that 12-lipoxygenase (12-LOX) may play an essential role in regulating apoptosis in rat Walker carcinosarcoma (W256) cells. Down regulation of 12-LOX gene expression by antisense oligonucleotides triggered time and dose dependent apoptosis. The W256 cell death, triggered by antisense oligos could be partially blocked by exogenous 12(S)-HETE. The antisense oligo treatment also resulted in a significant decrease in the ratio of Bcl-2/Bax proteins which are critical determinants of apoptosis. As expected, over expression of Bcl-2 protein provided partial death sparing effects (3). It also was demonstrated that exogenous arachidonic acid (AA) protected against apoptosis (4). Exogenous AA in a time and dose dependent fashion suppressed NDGA induced W256 apoptosis as well as DNA fragmentation. Serum withdrawal also caused W256 cells to undergo typical apoptosis which was not rescued by several growth factors commonly found in serum. However, exogenous AA suppressed serum starvation induced W256 cell apoptosis (4). Further studies demonstrated that the factor responsible for AA induced protection against apoptosis was the 12-LOX metabolite of AA 12(S)-HETE. 12-LOX has been demonstrated by our laboratory to be expressed in numerous tumor cells from human, rat and mouse (5). Factors controlling the regulation of 12-LOX are largely unknown. Several growth factors have been shown to stimulate 12-LOX activity in tumor cells. For example, in A431, human epidermoid carcinoma cells platelet type 12-LOX is expressed (6). Serum starvation induces a time and dose dependent reduction in the expression of 12-LOX protein (8). Another cytokine which has been demonstrated to regulate 12-LOX levels in tumor cells is autocrine motility factor (AMF). AMF stimulates cell motility and invasion via receptor mediated signaling pathways. AMF is also a potent growth factor. Our laboratory demonstrated that AMF induced cell motility was mediated by the lipoxygenase generation of 12(S)-HETE (7). In a subsequent report, we also demonstrated that AMF was able to increase the cellular protein levels of 12-LOX in high but not in low metastatic clones (8).

Publisher Summary This chapter discusses apoptosis and prostate cancer (PCa). Apoptosis, or programmed cell death, is characterized by distinct morphological (cell shrinkage, membrane blebbing, pyknosis, chromatin margination, cytoplasm contraction, and condensation) and biochemical features. Although apoptosis is considered a genetically regulated process as seen in physiologic events such as the involution of the mammary gland, it can be triggered, especially in in-vitro cultured cells, by physical and toxic exposures, cellular effects of hormones, growth factors, and cytokines, viral infection, and immunologic mechanisms. Example of an apoptosis regulator is p53, a phosphoprotein known to modulate gene transcription, police cell-cycle checkpoints, control DNA replication and repair, and maintain genomic stability. Wild-type p53 also positively regulates apoptosis. As apoptosis plays a critical role in the multiple steps (transformation, progression, and survival of metastases) of tumorigenesis, many chemoprevention and therapeutic regimens attempting to manipulate the apoptotic process have been proposed to aid in the clinical treatment of cancer patients. Apoptosis is closely involved in the initiation, progression, and metastasis of human PCa. The prostate, as an organ, is very similar to the breast of females in that both organs depend on circulating hormones for their growth. Therefore, androgens play a paramount role in regulating the growth of normal and malignant prostate epithelial cells.

Cancer metastasis is a complicated process. For a transformed cell to form a successful metastatic colony, it must in general complete all or most of the well-defined steps that comprise the “metastatic cascade”.1–4 The first step is uncontrolled cell proliferation, characteristic of both benign and malignant tumor cells. Intrinsic or acquired genetic instability, together with various epigenetic factors, generate tumor cell variants that acquire unique phenotypic characteristics that dissociate them from the parent tumor population and thus allow these variants to escape from the “social” constraints imposed by the host. This step confers on these “mutated” tumor cells invasive or metastatic capabilities and is generally considered to be the first step leading to site-specific metastasis. In the next step, tumor cells, in response to various chemoattractants and cytokines derived from the host and/or tumor cells, migrate towards neighboring vasculature or intravasate into the vasculature of the tumor and thus enter the hematogenous or lymphatic circulation. Subsequently, tumor cells travel to and arrest in the microcirculation by specific adherence to the endothelial cells of the target organ. Thereafter, tumor cells induce endothelial cell retraction, exit from circulation (extravasation), interact with the organ-specific extracellular matrix (ECM), proliferate in response to local (“soil”) growth factors, and finally form a metastatic colony. Failure at any one of these steps generally will abort the metastatic process. Completion of every step of the metastatic cascade is subject to a multitude of variable influences, an apparent example being the requirement of angiogenesis for the growth of both primary and secondary tumors.5.6

We have investigated the regulatory role of PGI2 and its stable analogs, i.e., iloprost and cicaprost, on 12(S)-HETE- and TPA-enhanced tumor cell integrin expression and adhesion. Walker 256 carcinosarcoma cells express alpha IIb beta 3 integrin receptors, which mediate their adhesion to endothelium, subendothelial matrix and fibronectin. Adhesion is enhanced by treatment with exogenous 12(S)-HETE but not 12(R)-HETE or other lipoxygenase-derived hydroxy fatty acids, as well as by TPA. Both 12(S)-HETE and TPA enhanced alpha IIb beta 3 expression on W256 cells. PGI2 iloprost and cicaprost inhibited both 12(S)-HETE- and TPA-enhanced adhesion to endothelium and subendothelial matrix as well as alpha IIb beta 3 expression on W256 cells. The mechanism responsible for the effect of PGI2 was explored. Prostacyclin treatment of W256 cells resulted in an enhanced production of cAMP in a time- and dose-dependent manner. Pre-treatment of tumor cells with increasing concentrations of adenosine resulted in a dose-dependent decrease in the PGI2 effect on TPA or 12(S)-HETE-enhanced adhesion, suggesting that the PGI2 effect is mediated through PKA. Dibutyryl cAMP also blocked the 12(S)-HETE- or TPA-enhanced adhesion, and adenosine pre-treatment did not result in an inhibition of the dibutyryl cAMP effect. Collectively, our results suggest that the cyclooxygenase metabolite PGI2 can antagonize the lipoxygenase metabolite 12(S)-HETE- and TPA-enhanced alpha IIb beta 3 expression and tumor cell adhesion via activation of adenylate cyclase and elevation of intracellular levels of cAMP.

Work functions and magnetic moments of C adsorbed Cr/Fe(001) surfaces with different C coverages θ and magnetic alignments (parallel or antiparallel) between Cr and Fe atom moments are investigated using first-principles methods based on density functional theory. The calculated results reveal that the spin configuration plays a significant role in determining the work function of the systems. The work functions of the systems with parallel states are evidently larger than those with antiparallel states. Moreover, for θ≤0.5 ML, with increasing value of θ, the work function increases from 4.23 eV to 5.13 eV for antiparallel states and from 4.47 eV to 5.44 eV for parallel states. While for θ>0.5 ML, the work function decreases with increasing value of θ. It can be also found that, for θ≤0.5 ML, the smaller the Cr and Fe magnetic moments are, the lower the Fermi energy EF is and the larger the work functions of the systems are. Based on analysis and discussion, we conclude that the changes of the work functions and magnetic moments are mainly determined by those of Fermi level and density of state (DOS) induced by the spin polarization, the electron transfer and the surface structure. Our work strongly suggests that controlling the magnetic states is a promising way for modulating the work function of magnetic metal gate.

This study has the potential to establish a new paradigm in adhesion-mediated control of eicosanoid production and could be relevant to tumor migration, invasion, and metastasis. 12-Lipoxygenase metabolizes arachidonic acid to form 12(S)-HETE, a metabolite known to play a significant role in the process of tumor-induced angiogenesis and metastasis. The role of 12-LOX in proliferation, survival, and motility of tumor cells as well as in angiogenesis has been described for a variety of cancers. Similarly Alpha6Beta4 integrin also plays a role in both angiogenesis and tumorigenesis in addition to having a role in the formation of hemidesmosomes (HD). Evidence suggests a correlation between 12-LOX and integrin Beta4 since they play a similar role in cancer progression. The physical interaction between 12-LOX and Beta4 was first identified by a yeast two-hybrid screen using 12-LOX as bait against a library made from A431 cells that are known to have endogenous expression of both proteins. We verified this interaction by co-immunoprecipitation from A431 cells, and recapitulated it in CHO and PC-3 cells ectopically expressing 12-LOX and the cytoplasmic tail of Beta4 integrin. Additionally our data shows that ligation or clustering of Alpha6Beta4 integrin by the natural ligand laminin or an antibody mimic of laminin, 3E1, caused an increase in translocation of 12-LOX from the cytosol to membrane and an increase in 12-LOX enzymatic activities as assayed by immunofluorescence and 12(S)-HETE measurements respectively. 3E1-stimulated A431 cells showed increased cell migration but there was 40% decrease in presence of 12-LOX inhibitor BMD122 which confirms the role of 12-LOX in cell invasion. Here we describe for the first time the interaction domains that unite Beta4 integrin and 12-LOX. We mapped the domains in the cytoplasmic tail of Beta4 critical for interacting with 12-LOX and found that the 1126-1157 domain in the cytoplasmic tail of Beta4 is important for binding of 12-LOX. Also we have shown the mutant fragment (1126-1315) interacts with 12-lipoxygenase in the cytosol and prevent its binding to native, full-length membrane-associated Beta4 and thereby prevents 12-LOX activation. This physical interaction has functional implications with regard to decreased cell survival and migration. Determination of the structural basis for protein-protein interaction may provide potential means to manipulate or block such interaction, which may yield therapeutic benefits. We propose that the results obtained from these studies will provide a novel, and important dimension to both integrin and eicosanoid biology. Acknowledgements: This project is supported by NIH: NCI, 2 R01 CA029997-17A1 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2890. doi:10.1158/1538-7445.AM2011-2890

12-Lipoxygenase (12-LOX) converts arachidonic acid to 12(S)-hydroxyeicosatetraenoic acid 12(S)-HETE, an eicosanoid implicated in tumor angiogenesis, growth, and metastasis. We have demonstrated previously that 12-LOX interacts with integrin beta4 cytoplasmic domain by using yeast two hybrid system. Integrins are involved in metastasis by mediating attachment and migration of cells, as well as through transducing signals. Increased expression of 12-LOX and beta4 integrin have been documented in a number of carcinomas. The purpose of this study is to find out the mechanisms underlying the signaling pathways in 12-LOX mediated cancer progression. The phosphorylation of beta4 integrin, Src, and 12-LOX were analyzed using specific antibodies against phospho-tyrosine, phospho-beta4 integrin, Phospho-Src (pSrc). c-Src activity and association was determined in beta4 integrin immunoprecipitate. The HEK293 cells transiently transfected with wild type or dominant negative (DN) Src were used as in vitro models to study the regulation of 12-LOX activity. The 12-LOX product 12(S)-HETE was analyzed by using an analytical liquid chromatography-tandem mass spectrometry.12-LOX tyrosine phosphorylation sites mutants viz., 19, 295, and 614 were used to study the effect on its activity upon beta4 ligation. We show here that the cytoplasmic domain of beta4 integrin, can interact specifically with pSrc after beta4 integrin ligation in A431 cells. Herein we report that beta4 integrin ligation resulted in significant increase in 12(S)-HETE level in A431 cells, compared to that of unligated beta4 integrin. An increase in Src kinase activity was found in beta4 integrin ligation of A431 cells and inhibition of Src kinase by PP2 or DN Src significantly reduced 12(S)-HETE production. The ligation of beta4 integrin induces src activation, which resulted in tyrosine phosphorylation of 12-LOX. The residues that are important for 12-LOX activity and tyrosine phosphorylation were mapped to Y19 and Y614. We examined that 12-LOX mutants (Y19F and Y614F), showed 70% less enzymatic activity, compared to wild type 12-LOX. Furthermore, we showed that the 12-LOX activity modulated by these residues impacts migration in HUVEC cells. To our knowledge, this is the first report to show that Src kinase activity is required for beta4 integrin mediated regulation of 12-LOX, which may in turn regulate metastatic potential of cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2902. doi:10.1158/1538-7445.AM2011-2902

Background Integrins and enzymes of the eicosanoid pathway are both well-established contributors to cancer. However, this is the first report of the interdependence of the two signaling systems. In a screen for proteins that interacted with, and thereby potentially regulated, the human platelet-type 12-lipoxygenase (12-LOX, ALOX12), we identified the integrin β4 (ITGB4). Methods Using a cultured mammalian cell model, we have demonstrated that ITGB4 stimulation leads to recruitment of 12-LOX from the cytosol to the membrane where it physically interacts with the integrin to become enzymatically active to produce 12(S)-HETE, a known bioactive lipid metabolite that regulates numerous cancer phenotypes. Results The net effect of the interaction was the prevention of cell death in response to starvation. Additionally, regulation of β4-mediated, EGF-stimulated invasion was shown to be dependent on 12-LOX, and downstream Erk signaling in response to ITGB4 activation also required 12-LOX. Conclusions This is the first report of an enzyme of the eicosanoid pathway being recruited to and regulated by activated β4 integrin. Integrin β4 has recently been shown to induce expansion of prostate tumor progenitors and there is a strong correlation between stage/grade of prostate cancer and 12-LOX expression. The 12-LOX enzymatic product, 12(S)-HETE, regulates angiogenesis and cell migration in many cancer types. Therefore, disruption of integrin β4-12LOX interaction could reduce the pro-inflammatory oncogenic activity of 12-LOX. This report on the consequences of 12-LOX and ITGB4 interaction sets a precedent for the linkage of integrin and eicosanoid biology through direct protein-protein association. Electronic supplementary material The online version of this article (doi:10.1186/s12943-015-0382-5) contains supplementary material, which is available to authorized users.