SAP deletion promotes malignant insulinoma progression by inducing CXCL12 secretion from CAFs via the CXCR4/p38/ERK signalling pathway.

Malignant insulinoma is an extremely rare type of functioning pancreatic neuroendocrine tumour with a high degree of malignancy and a high incidence of metastasis. However, it is still unclear how malignant insulinomas develop and metastasize. Serum amyloid P component (SAP), a member of the pentraxin protein family, is an acute-phase protein secreted by liver cells. The role of SAP in insulinoma and the related mechanism are still unknown. To determine the effect of SAP on insulinoma, we crossed Rip1-Tag2 mice, which spontaneously develop insulinoma, and SAP knockout (KO) mice to generate Rip1-Tag2;SAP-/- mice. We found that SAP deletion significantly promoted the growth, invasion and metastasis of malignant insulinoma through C-X-C motif chemokine ligand 12 (CXCL12) secreted by cancer-associated fibroblasts (CAFs). Further study showed that SAP deletion promoted CXCL12 secretion by CAFs through the CXCR4/p38/ERK signalling pathway. These findings reveal a novel role and mechanism of SAP in malignant insulinoma and provide direct evidence that SAP may be a therapeutic agent for this disease.

NF-κB Activator 1 downregulation in macrophages activates STAT3 to promote adenoma-adenocarcinoma transition and immunosuppression in colorectal cancer.

BACKGROUND: Adenoma-adenocarcinoma transition is a key feature of colorectal cancer (CRC) occurrence and is closely regulated by tumor-associated macrophages (TAMs) and CD8+ T cells. Here, we investigated the effect of the NF-κB activator 1 (Act1) downregulation of macrophages in the adenoma-adenocarcinoma transition. METHODS: This study used spontaneous adenoma-developing ApcMin/+, macrophage-specific Act1-knockdown (anti-Act1), and ApcMin/+; anti-Act1 (AA) mice. Histological analysis was performed on CRC tissues of patients and mice. CRC patients' data retrieved from the TCGA dataset were analyzed. Primary cell isolation, co-culture system, RNA-seq, and fluorescence-activated cell sorting (FACS) were used. RESULTS: By TCGA and TISIDB analysis, the downregulation of Act1 expression in tumor tissues of CRC patients negatively correlated with accumulated CD68+ macrophages in the tumor. Relative expression of EMT markers in the tumor enriched ACT1lowCD68+ macrophages of CRC patients. AA mice showed adenoma-adenocarcinoma transition, TAMs recruitment, and CD8+ T cell infiltration in the tumor. Macrophages depletion in AA mice reversed adenocarcinoma, reduced tumor amounts, and suppressed CD8+ T cell infiltration. Besides, macrophage depletion or anti-CD8a effectively inhibited metastatic nodules in the lung metastasis mouse model of anti-Act1 mice. CRC cells induced activation of IL-6/STAT3 and IFN-γ/NF-κB signaling and the expressions of CXCL9/10, IL-6, and PD-L1 in anti-Act1 macrophages. Anti-Act1 macrophages facilitated epithelial-mesenchymal-transition and CRC cells' migration via CXCL9/10-CXCR3-axis. Furthermore, anti-Act1 macrophages promoted exhaustive PD1+ Tim3+ CD8+ T cell formation. Anti-PD-L1 treatment repressed adenoma-adenocarcinoma transition in AA mice. Silencing STAT3 in anti-Act1 macrophages reduced CXCL9/10 and PD-L1 expression and correspondingly inhibited epithelial-mesenchymal-transition and CRC cells' migration. CONCLUSIONS: Act1 downregulation in macrophages activates STAT3 that promotes adenoma-adenocarcinoma transition via CXCL9/10-CXCR3-axis in CRC cells and PD-1/PD-L1-axis in CD8+ T cells.

CYD0281, a Bcl-2 BH4 domain antagonist, inhibits tumor angiogenesis and breast cancer tumor growth.

BACKGROUND: B-cell lymphoma 2 (Bcl-2) family proteins are key regulators of apoptosis, which possess four conserved Bcl-2 homologies (BH) domains. Among the BH domains, the BH3 domain is considered as a potent 'death domain' while the BH4 domain is required for anti-apoptotic activity. Bcl-2 can be converted to a pro-apoptotic molecule through the removal or mutation of the BH4 domain. Bcl-2 is considered as an inducer of angiogenesis, which can promote tumor vascular network formation and further afford nutrients and oxygen to promote tumor progression. However, whether disrupting the function of the BH4 domain to convert Bcl-2 into a pro-apoptotic molecule could make Bcl-2 possess the potential for anti-angiogenic therapy remains to be defined. METHODS: CYD0281 was designed and synthesized according to the lead structure of BDA-366, and its function on inducing a conformational change of Bcl-2 was further evaluated via immunoprecipitation (IP) and immunofluorescence (IF) assays. Moreover, the function of CYD0281 on apoptosis of endothelial cells was analyzed via cell viability, flow cytometry, and western blotting assays. Additionally, the role of CYD0281 on angiogenesis in vitro was determined via endothelial cell migration and tube formation assays and rat aortic ring assay. Chick embryo chorioallantoic membrane (CAM) and yolk sac membrane (YSM) models, breast cancer cell xenograft tumor on CAM and in mouse models as well as the Matrigel plug angiogenesis assay were used to explore the effects of CYD0281 on angiogenesis in vivo. RESULTS: We identified a novel potent small-molecule Bcl-2-BH4 domain antagonist, CYD0281, which exhibited significant anti-angiogenic effects both in vitro and in vivo, and further inhibited breast cancer tumor growth. CYD0281 was found to induce conformational changes in Bcl-2 through the exposure of the BH3 domain and convert Bcl-2 from an anti-apoptotic molecule into a cell death inducer, thereby resulting in the apoptosis of vascular endothelial cells. CONCLUSIONS: This study has revealed CYD0281 as a novel Bcl-2-BH4 antagonist that induces conformational changes of Bcl-2 to convert to a pro-apoptotic molecule. Our findings indicate that CYD0281 plays a crucial role in anti-angiogenesis and may be further developed as a potential anti-tumor drug candidate for breast cancer. This work also provides a potential anti-angiogenic strategy for breast cancer treatment.

Fe-Catalyzed Radical Trifluoromethyl-Alkenylation of Alkenes or Alkynes with 2-Amino-1,4-naphthoquinones.

The first Fe-catalyzed three-component radical trifluoromethyl-alkenylation of alkenes with 2-amino-1,4-naphthoquinones and CF3SO2Na is reported. The developed reaction enables the highly regioselective preparation of a variety of valuable CF3-substituted 1,4-naphthoquinones in acceptable yields. In the light of the catalytic system, alkynes smoothly afford the corresponding three- or four-component trifluoromethyl-alkenylation products. This protocol features use of easily available and inexpensive reagents, broad substrate scope, and simple reaction conditions.

Quantum mechanics/molecular mechanics studies on excited state decay pathways of 5-azacytosine in aqueous solution.

In this work, we have used the QM(CASPT2//CASSCF)/MM approach to study the photophysical properties and relaxation mechanism of 5-azacytosine (5-AC) in aqueous solution. Based on the relevant minimum-energy structures and intersection structures, and excited-state decay paths in the S1, S2, T1, T2, and S0 states, several feasible excited-state nonradiative decay channels from the initially populated S2(ππ*) state are proposed. Two major channels are singlet-mediated nonradiative pathways, in which the S2 system will internally convert (IC) to the S0 state directly or mediated by the 1nπ* state via a 1ππ*/1nπ* conical intersection. The minor ones are related to intersystem crossing (ISC) processes. The system would populate to the T1 state via the S2 → S1 → T1 or S2 → T2 → T1 ISC process, followed by further decay to the S0 state via the transition from T1 to S0. However, due to small spin-orbit couplings (SOCs) at the singlet-triplet crossing points, the related ISC would be less efficient and probably take longer. The present work rationalizes the ultrafast excited-state decay dynamics of 5-AC in aqueous solution and its low quantum yields of triplets and fluorescence. It provides important mechanistic insights into understanding 5-AC's derivatives and analogues.

R5, a neutralizing antibody to Robo1, suppresses breast cancer growth and metastasis by inhibiting angiogenesis via down-regulating filamin A.

Breast cancer (BC) is one of the most common cancers among women in both developed and developing countries with a rising incidence. Using the MMTV-PyMT transgenic mouse model and xenografted breast cancer model, we found that R5, a neutralizing antibody to Robo1, significantly inhibited BC growth and metastasis. Angiogenesis is involved in the growth and metastasis of BC. Interestingly, R5 significantly decreases microvessel density in BC tissues, and inhibits blood vessel formation and development in in vivo chick embryo chorioallantoic membrane (CAM), yolk sac membrane (YSM) and Matrigel plug models. To investigate whether its anti-breast cancer efficacy is ascribed to its direct antiangiogenic properties, xenografted breast cancer model on CAM was established. Furthermore, R5 significantly reduces the tube formation of the vascular plexus on xenografted breast tumor on CAM. R5 also suppresses the migration and the tubular structure formation of human umbilical vein endothelial cells (HUVECs) by down-regulating the expression of filamin A (FLNA). These findings show that R5 has the potential to be a promising agent for the treatment of BC by suppressing the tumor-induced angiogenesis.

Decylubiquinone suppresses breast cancer growth and metastasis by inhibiting angiogenesis via the ROS/p53/ BAI1 signaling pathway.

Breast cancer is one of the most common cancers worldwide with a rising incidence, and is the leading cause of cancer-related death among females. Angiogenesis plays an important role in breast cancer growth and metastasis. In this study, we identify decylubiquinone (DUb), a coenzyme Q10 analog, as a promising anti-breast cancer agent through suppressing tumor-induced angiogenesis. We screened a library comprising FDA-approved drugs and found that DUb significantly inhibits blood vessel formation using in vivo chick embryo chorioallantoic membrane (CAM) and yolk sac membrane (YSM) models. DUb was further identified to inhibit angiogenesis in the rat aortic ring and Matrigel plug assay. Moreover, DUb was found to suppress breast cancer growth and metastasis in the MMTV-PyMT transgenic mouse and human xenograft tumor models. To explore whether the anticancer efficacy of DUb was directly corrected with tumor-induced angiogenesis, the MDA-MB-231 breast cancer assay on the CAM was performed. Interestingly, DUb significantly inhibits the angiogenesis of breast cancer on the CAM. Brain angiogenesis inhibitor 1 (BAI1), a member of the G protein-coupled receptor (GPCR) adhesion subfamily, has an important effect on the inhibition of angiogenesis. Further studies demonstrate that DUb suppresses the formation of tubular structures by regulating the reactive oxygen species (ROS)/p53/BAI1 signaling pathway. These results uncover a novel finding that DUb has the potential to be an effective agent for the treatment of breast cancer by inhibiting tumor-induced angiogenesis.

Insights into Substrate Self-Assisted Activation of Allylic Alcohols Guiding to Mild Allylic Substitution of Tautomerizable Heteroarenes.

A substrate self-assisted activation of allylic alcohols by tautomerizable heteroarenes via hydrogen bonding was disclosed by various NMR techniques, including variable-temperature 1H NMR, Job plot, and 1H NMR titration. Guided by these finding, a much milder allylic substitution of tautomerizable heteroarenes with allylic alcohols was developed, affording the target products in high yields.

Increased Expression of IL-23 and IL-17 in Serum of Patients with Neonatal Respiratory Distress Syndrome and its Clinical Significance.

BACKGROUND: The present study focused on the potential clinical significance of Th-17 cell related inflammatory cytokines in the occurrence and development of neonatal respiratory distress syndrome (NRDS). METHODS: We included 82 NRDS children and 82 healthy controls. NRDS children were divided into the mild and severe group based on the disease severity. The serum samples of the NRDS and non-NRDS children were collected, and the expression levels of IL-17, IL-22, and IL-23 were determined by ELISA method. Moreover, correlation between the levels of the cytokines and the disease severity were analyzed, and receiver operating characteristics curve (ROC) analysis was performed to determine the diagnostic value of the cytokines. Finally, correlation between the lung ultrasound score (LUS) of the NRDS patients and the levels of IL-17 and IL-23 were analyzed. RESULTS: IL-17 and IL-23 were dramatically increased in serum of the NRDS patients compared with the non-NRDS patients; moreover, IL-17 and IL-23 were significantly higher in the severe compared with the mild NRDS group, and the levels of both IL-17 and IL-23 were positively correlated with the disease severity. Furthermore, ROC analysis showed that both IL-17 and IL-23 can distinguish NRDS patient, especially the severe NRDS patients from the non-NRDS patients with high sensitivity and specificity; finally, the levels of IL-17 and IL-23 were positively correlated with the LUS in NRDS patients. CONCLUSIONS: IL-17 and IL-23 were up-regulated in NRDS and may serve as sensitive biomarkers for the diagnosis and treatment of the disease.

Substrate Self-Assisted Secondary Bond Activation of Allylic Alcohol in a Tsuji-Trost Reaction Revealed by NMR Methods.

The first experimental evidence for the palladium-catalyzed secondary bond activation of allylic alcohols in a Tsuji-Trost reaction was provided by NMR methods, such as variable-temperature 1H NMR, diffusion-ordered spectroscopy (DOSY), Job's method, 1H NMR titration, and nuclear Overhauser enhancement spectroscopy (NOESY). The experimental results revealed that the substrate self-assisted activation of allylic alcohols is probably performed via a 1:1 binding six-membered-ring complex, which are formed by the formation of the secondary bonds, the hydrogen bond and P···O noncovalent bond between allylic alcohol and phosphonium ylide.

Li-N2 Batteries: A Reversible Energy Storage System?

Tremendous energy consumption is required for traditional artificial N2 fixation, leading to additional environmental pollution. Recently, new Li-N2 batteries have inextricably integrated energy storage with N2 fixation. In this work, graphene is introduced into Li-N2 batteries and enhances the cycling stability. However, the instability and hygroscopicity of the discharge product Li3 N lead to a rechargeable but irreversible system. Moreover, strong nonpolar N≡N covalent triple bonds with high ionization energies also cause low efficiency and irreversibility of Li-N2 batteries. In contrast, the modification with in situ generated Li3 N and LiOH restrained the loss and volume change of Li metal anodes during stripping and plating, thereby promoting the rechargeability of the Li-N2 batteries. The mechanistic study here will assist in the design of more stable Li-N2 batteries and create more versatile methods for N2 fixation.

EndophilinA2 protects against angiotensin II-induced cardiac hypertrophy by inhibiting angiotensin II type 1 receptor trafficking in neonatal rat cardiomyocytes.

Cardiac hypertrophy is one of the major risk factors for chronic heart failure. The role of endophilinA2 (EndoA2) in clathrin-mediated endocytosis and clathrin-independent endocytosis is well documented. In the present study, we tested the hypothesis that EndoA2 protects against angiotensin II (Ang II)-induced cardiac hypertrophy by mediating intracellular angiotensin II type 1 receptor (AT1-R) trafficking in neonatal rat cardiomyocytes (NRCMs). Cardiac hypertrophy was evaluated by using cell surface area and quantitative RT-PCR (qPCR) analyses. For the first time, we found that EndoA2 attenuated cardiac hypertrophy and fibrosis induced by Ang II. Moreover, EndoA2 inhibited apoptosis induced by excessive endoplasmic reticulum stress (ERS), which accounted for the beneficial effects of EndoA2 on cardiac hypertrophy. We further revealed that there was an interaction between EndoA2 and AT1-R.The expression levels of EndoA2, which inhibits AT1-R transport from the cytoplasm to the membrane, and the interaction between EndoA2 and AT1-R were obviously decreased after Ang II treatment. Furthermore, Ang II inhibited the co-localization of AT1-R with GRP-78, which was reversed by EndoA2 overexpression. In conclusion, our results suggested that EndoA2 plays a role in protecting against cardiac hypertrophy induced by Ang II, possibly by inhibiting AT1-R transport from the cytoplasm to the membrane to suppress signal transduction.

Disruption of the Igf2 gene alters hepatic lipid homeostasis and gene expression in the newborn mouse.

Newborns with intrauterine growth-restriction are at increased risk of mortality and life-long comorbidities. Insulin-like growth factor-II (IGF2) deficiency in humans, as well as in mice, leads to intrauterine growth restriction and decreased neonatal glycogen stores. The present study aims to further characterize the metabolic and transcriptional consequences of Igf2 deficiency in the newborn. We found that, despite being born significantly smaller than their wild-type ( Igf2+/+) littermates, brain size was preserved in Igf2 knockout ( Igf2-/-), consistent with nutritional deficiency. Histological and triglyceride analyses of newborn livers revealed that Igf2-/- mice are born with hepatic steatosis. Gene expression analysis in Igf2-/- newborn livers showed an alteration of genes known to be dysregulated in chronic caloric restriction, including the most upregulated gene, serine dehydratase. Multiple genes connected with lipid metabolism and/or hepatic steatosis were also upregulated. Ingenuity Pathway Analysis confirmed that the biological functions most altered in livers of Igf2-/- newborns are related to lipid metabolism, with the top upstream regulator predicted to be the peroxisome proliferator-activated receptor alpha, a master regulator of hepatic lipid and carbohydrate homeostasis. Together, our data indicate that Igf2 deficiency leads to a newborn phenotype strongly reminiscent of nutritional deficiency, including growth retardation, increased brain/body weight ratio, hepatic steatosis, and characteristic changes in hepatic gene expression. We propose that in addition to its growth factor proliferating functions, Igf2 may also regulate growth by altering the expression of genes that control nutrient metabolism in the newborn.

Inactivation of PI3Kδ induces vascular injury and promotes aneurysm development by upregulating the AP-1/MMP-12 pathway in macrophages.

OBJECTIVE: An aneurysm is an inflammatory vascular condition. Phosphatidylinositol 3-kinases δ is highly expressed in leukocytes, and play a key role in innate immunity. However, the link between phosphatidylinositol 3-kinases δ and aneurysm development has not yet been elucidated. APPROACH AND RESULTS: Carotid ligation unexpectedly induced characteristic aneurysm formation beneath the ligation point in p110δ(D910A/D910A) mice (n=25; P<0.001 versus wild-type). Besides, p110δ inactivation exacerbated CaCl2-induced abdominal aortic aneurysms development. A reverse transcription polymerase chain reaction microarray revealed significant extracellular matrix components degradation and matrix metalloproteinases (MMPs) upregulation in the abdominal aorta of p110δ(D910A/D910A) mice. Similarly, the expression of both collagen I and IV was significantly decreased (n=10; P<0.05 versus wild-type) in carotid artery. Western blot assay confirmed that MMP-12 was significantly upregulated in arteries of p110δ(D910A/D910A) mice (n=10; P<0.01 versus wild-type). In vitro, p110δ inactivation marked increase peritoneal macrophages recruitment and synergistically enhance tumor necrosis factor-α-induced recruitment. A specific phosphatidylinositol 3-kinases δ inhibitor (IC87114) or genetic p110δ inactivation upregulated MMP-12 expression and c-Jun phosphorylation (n=6; P<0.05 versus wild-type macrophages). IC87114 also increased activator protein-1 DNA-binding activity (n=6; P<0.001 versus control) and enhanced the effect of tumor necrosis factor-α on activator protein-1-binding activity (n=5; P<0.01 versus tumor necrosis factor-α treatment groups). Knockdown of c-Jun suppressed the effect of the IC87114 and tumor necrosis factor-α on MMP-12 mRNA expression (n=5 in each group; P<0.01 versus scrRNA treatment groups). CONCLUSIONS: Our findings demonstrate that p110δ inactivation leads to extracellular matrix degradation in vessels and promotes aneurysm development by inducing macrophages migration and upregulating the activator protein-1/MMP-12 pathway in macrophages.

Activation of Slit2-Robo1 signaling promotes liver fibrosis.

BACKGROUND & AIMS: The secretory protein Slit2 and its receptor Robo1 are believed to regulate cell growth and migration. Here, we aimed to determine whether Slit2-Robo1 signaling mediates the pathogenesis of liver fibrosis. METHODS: Serum levels of Slit2 in patients with liver fibrosis were determined by ELISA. Liver fibrosis was induced in wild-type (WT), Slit2 transgenic (Slit2-Tg) and Robo1(+/-)Robo2(+/-) double heterozygotes (Robo1/2(+/-)) mice by carbon tetrachloride (CCl4). The functional contributions of Slit2-Robo1 signaling in liver fibrosis and activation of hepatic stellate cells (HSCs) were investigated using primary mouse HSCs and human HSC cell line LX-2. RESULTS: Significantly increased serum Slit2 levels and hepatic expression of Slit2 and Robo1 were observed in patients with liver fibrosis. Compared to WT mice, Slit2-Tg mice were much more vulnerable to CCl4-induced liver injury and more readily develop liver fibrosis. Development of hepatic fibrosis in Slit2-Tg mice was associated with a stronger hepatic expression of collagen I and α-smooth muscle actin (α-SMA). However, liver injury and hepatic expression of collagen I and α-SMA were attenuated in CCl4-treated Robo1/2(+/-) mice in response to CCl4 exposure. In vitro, Robo1 neutralizing antibody R5 and Robo1 siRNA downregulated phosphorylation of Smad2, Smad3, PI3K, and AKT in HSCs independent of TGF-ß1. R5 and Robo1 siRNA also inhibited the expression of α-SMA by HSCs. Finally, the protective effect of R5 on the CCl4-induced liver injury and fibrosis was further verified in mice. CONCLUSIONS: Slit2-Robo1 signaling promotes liver injury and fibrosis through activation of HSCs.

Effects of high fructose and salt feeding on systematic metabonome probed via (1) H NMR spectroscopy.

Diets rich in high fructose and salt are increasingly popular in our daily life. A combination consumption of excessive fructose and salt can induce insulin resistance (IR) and hypertension (HT), which are major public health problems around the world. However, the effects of high fructose and salt on systematic metabonome remain unknown, which is very important for revealing the molecular mechanism of IR and HT induced by this dietary pattern. The metabolic profiling in urine, plasma, and fecal extracts from high fructose and salt-fed rats was investigated by use of (1) H nuclear magnetic resonance (NMR)-based metabonomics approach in this study. Multivariate analysis of NMR data showed the effects of high fructose and salt on the global metabonome. The metabolite analysis in urine and fecal extracts showed the time-dependent metabolic changes, which displayed metabonomic progression axes from normal to IR and HT status. The changes of 2-oxoglutarate, creatine and creatinine, citrate, hippurate, trimethylamine N-oxide (TMAO), and betaine in urine, together with gut microbiota disorder in feces, were observed at the preliminary formation stage of IR and HT (fourth week). At the severe stage (eighth week), the previously mentioned metabolic changes were aggravated, and the changes of lipid and choline metabolism in plasma suggested the increased risk of cardiovascular diseases. These findings provide an overview of biochemistry consequences of high fructose and salt feeding and comprehensive insights into the progression of systematic metabonome for IR and HT induced by this dietary pattern.

EPAC inhibitor suppresses angiogenesis and tumor growth of triple-negative breast cancer.

AIMS: Exchange protein directly activated by cAMP 1 (EPAC1), a major isoform of guanine nucleotide exchange factors, is highly expressed in vascular endothelia cells and regulates angiogenesis in the retina. High intratumor microvascular densities (MVD) resulting from angiogenesis is responsible for breast cancer development. Downregulation of EPAC1 in tumor cell reduces triple-negative breast cancer (TNBC)-induced angiogenesis. However, whether Epac1 expressed in vascular endothelial cells contributes to angiogenesis and tumor development of TNBC remains elusive. MAIN METHODS: We employed NY0123, a previously identified potent EPAC inhibitor, to explore the anti-angiogenic biological role of EPAC1 in vitro and in vivo through vascular endothelial cells, rat aortic ring, Matrigel plug, and chick embryo chorioallantoic membrane (CAM) and yolk sac membrane (YSM) assays, as well as the in vivo xenograft tumor models of TNBC in both chick embryo and mice. KEY FINDINGS: Inhibiting EPAC1 in vascular endothelial cells by NY0123 significantly suppresses angiogenesis and tumor growth of TNBC. In addition, NY0123 possesses a better inhibitory efficacy than ESI-09, a reported specific EPAC inhibitor tool compound. Importantly, inhibiting EPAC1 in vascular endothelia cells regulates the typical angiogenic signaling network, which is associated with not only vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor-2 (VEGFR2) signaling, but also PI3K/AKT, MEK/ERK and Notch pathway. CONCLUSIONS: Our findings support that EPAC1 may serve as an effective anti-angiogenic therapeutic target of TNBC, and EPAC inhibitor NY0123 has the therapeutic potential to be developed for the treatment of TNBC.

Downregulation of TMEM16A calcium-activated chloride channel contributes to cerebrovascular remodeling during hypertension by promoting basilar smooth muscle cell proliferation.

BACKGROUND: The Ca(2+)-activated chloride channel (CaCC) plays an important role in a variety of physiological functions. In vascular smooth muscle cells, CaCC is involved in the regulation of agonist-stimulated contraction and myogenic tone. The physiological functions of CaCC in blood vessels are not fully revealed because of the lack of specific channel blockers and the uncertainty concerning its molecular identity. METHODS AND RESULTS: Whole-cell patch-clamp studies showed that knockdown of TMEM16A but not bestrophin-3 attenuated CaCC currents in rat basilar smooth muscle cells. The activity of CaCC in basilar smooth muscle cells isolated from 2-kidney, 2-clip renohypertensive rats was decreased, and CaCC activity was negatively correlated with blood pressure (n=25; P<0.0001) and medial cross-sectional area (n=24; P<0.0001) in basilar artery during hypertension. Both upregulation of CaMKII activity and downregulation of TMEM16A expression contributed to the reduction of CaCC in the hypertensive basilar artery. Western blot results demonstrated that angiotensin II repressed TMEM16A expression in basilar smooth muscle cells (n=6; P<0.01). Knockdown of TMEM16A facilitated and overexpression of TMEM16A inhibited angiotensin II-induced cell cycle transition and cell proliferation determined by flow cytometry and BrdU incorporation (n=6 in each group; P<0.05). TMEM16A affected cell cycle progression mainly through regulating the expression of cyclin D1 and cyclin E. CONCLUSIONS: TMEM16A CaCC is a negative regulator of cell proliferation. Downregulation of CaCC may play an important role in hypertension-induced cerebrovascular remodeling, suggesting that modification of the activity of CaCC may be a novel therapeutic strategy for hypertension-associated cardiovascular diseases such as stroke.

1H-NMR based metabolic study of MMTV-PyMT mice along with pathological progress to screen biomarkers for the early diagnosis of breast cancer.

A 1H NMR-based metabonomic approach was applied to monitor the alterations of serum metabolic profiles in MMTV-PyMT transgenic mice to detect the dynamic changes associated with the pathological process and explore the early-stage biomarkers. The 1H NMR spectra of sera samples from four different stages in MMTV-PyMT mice including hyperplasia, adenoma, early carcinoma and late carcinoma stages were recorded and analyzed using multivariate statistical techniques. The results showed that the increased levels of lipid and lactate, and decreased leucine/isoleucine, valine, methionine, glutamine, creatine, PC/GPC, taurine and glucose were of significance for the early carcinoma stage. As the disease progressed (late carcinoma stage), the metabolic profiles changed significantly; some were negatively regulated compared with those at the early carcinoma stage, such as lipid, leucine/isoleucine, methionine and creatine, accompanied by other new metabolite changes of alanine, pyruvate, glutamate, citrate, aspartate, myo-inositol, 3-methylhistidine and formate. It is important to note that breast cancer patients and the early carcinoma stage of MMTV-PyMT mice had some similar metabolite changes, including lipid, lactate, glutamine, creatine, taurine and glucose, which were determined to be of great value for the early clinical diagnosis of breast cancer. The findings from this study provided valuable biomarkers for the early clinical diagnosis of breast cancer, and showed the potential power of integrating NMR techniques and pattern recognition methods for the analysis of the biochemical changes under certain pathophysiological conditions.

Glipizide Combined with ANP Suppresses Breast Cancer Growth and Metastasis by Inhibiting Angiogenesis through VEGF/VEGFR2 Signaling.

BACKGROUND: Breast cancer is one of the most common cancers worldwide among women, and angiogenesis has an important effect on its growth and metastasis. Glipizide, which is a widely used drug for type 2 diabetes mellitus, has been reported to inhibit tumor growth and metastasis by upregulating the expression of natriuretic peptide receptor A (NPRA). Atrial natriuretic peptide (ANP), the receptor of NPRA, plays an important role in angiogenesis. The purpose of this study was to explore the effect of glipizide combined with ANP on breast cancer growth and metastasis. METHODS: This study aimed at investigating the effect of glipizide combined with ANP on breast cancer. Glipizide, ANP, or glipizide combined with ANP was intraperitoneally injected into MMTV-PyMT mice. To explore whether the anticancer efficacy of glipizide combined with ANP was correlated with angiogenesis, a tube formation assay was performed. RESULTS: Glipizide combined with ANP was found to inhibit breast cancer growth and metastasis in MMTV-PyMT mice, which spontaneously develop breast cancer. Furthermore, the inhibitory effect of ANP combined with glipizide was better than that of glipizide alone. ANP combined with glipizide significantly inhibited tube formation of human umbilical vein endothelial cells (HUVECs) by suppressing vascular endothelial growth factor (VEGF)/VEGFR2 (vascular endothelial growth factor receptor 2) signaling. CONCLUSION: These results demonstrate that glipizide combined with ANP has a greater potential than glipizide alone to be repurposed as an effective agent for the treatment of breast cancer by targeting tumor-induced angiogenesis.