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Aim: We investigated the direct effects of sevoflurane on angiogenesis and a variety of tumor cells. Materials & methods: The antiangiogenic activity of sevoflurane was determined using angiogenesis and biochemical assays. Results: Sevoflurane at low doses inhibits capillary network formation. Sevoflurane inhibited VEGF- and bFGF-stimulated migration, adhesion and growth in endothelial cells and induced apoptosis. Sevoflurane only at high doses inhibited growth and migration of tumor cells, suggesting differential effects of sevoflurane between endothelial and tumor cells. Mechanistically, sevoflurane decreased growth factors-induced Ras and Rac1 activation, and suppressed Ras and Rac1 signaling. Conclusion: We demonstrate the antiangiogenic effects of sevoflurane and provide preclinical evidence into the potential mechanisms by which sevoflurane may negatively affect cancer growth and metastasis.

Norcantharidin (NCTD), a demethylated derivative of cantharidin, has been reported to exhibit activity against various types of cancers. However, the anti-invasive effects of NCTD and its molecular mechanism in human mucoepidermoid carcinoma (MEC) remain incompletely elucidated. Clonogenic, wound healing, invasion, zymography, western blotting and immunocytochemistry assays were performed in YD-15 cells to investigate the anti-invasive effect of NCTD and its molecular mechanism of action. The inhibitory effects of NCTD on invasiveness were compared with those of a novel focal adhesion kinase (FAK) kinase inhibitor, PF-562271. NCTD markedly suppressed the colony formation, migration, and invasion of YD-15 cells as well as the activities of MMP-2 and MMP-9. It disrupted F-actin reorganization through suppressing the FAK/Paxillin axis. Moreover, NCTD exhibited a powerful anti-invasive effect compared with that of PF-562271 in YD-15 cells. Collectively, these results suggest that NCTD has a potential anti-invasive activity against YD-15 cells. This study may clarify the impact of NCTD on migration and invasion of human MEC cells.

Background: Bone metastases are a common feature of advanced prostatic malignancies. They are characterised by a unique prevalence of osteoblastic phenotype and a poor prognosis. Paxillin is a 68-kDa signal transduction adaptor and scaffold protein that contains motifs involved in the mediation of protein-protein interactions. The state of paxillin phosphorylation is central to determining a cell's ability to adhere, detach and migrate and hence has been linked to processes such as wound repair and tumour metastasis. The current study explored the impact of paxillin suppression on prostate and breast cancer cell function and their responsiveness to hepatocyte growth factor (HGF) and bone matrix extract (BME) in order to assess its potential to influence bone colonization and homing., Materials and Methods: Hammerhead ribozyme transgenes were used to knockdown the expression of paxillin in breast and prostate cancer cell lines. The impact on the cell growth, migration, adhesion and invasion was assessed using in vitro functional assays. In order to explore potential mechanisms, focal adhesion kinase (FAK) inhibitor was also used., Results: Knockdown of paxillin expression was observed in all tested cell lines following transfection with the ribozyme transgene. The knockdown of paxillin increased proliferation and invasiveness of LNCaP cells, with no effect on their attachment abilities. The opposite, however, is true for PC-3 cells where, following knockdown, cellular attachment was significantly reduced, while no significant changes in growth and invasiveness were detected. In the MDA-MB-231 breast cancer knockdown model, cells had little difference in proliferative rates and generally increased attachment and reduced invasive abilities. Treatments with HGF and BME had differential effects on targeted cells when compared to controls., Conclusion: These data suggest that paxillin appears to influence major cell functions in a diverse range of prostate and breast cancer models. The responsiveness of cells to environmental factors such as HGF or BME may be influenced by paxillin status, although this seems to be dependent on cell type., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Pancreatic ductal adenocarcinoma (PDAC) is a devastating cancer in large part due to late diagnosis and a lack of effective screening tests. In spite of recent progress in imaging, surgery and new therapeutic options for pancreatic cancer, the overall five-year survival still remains unacceptably low. Numerous studies have shown that focal adhesion kinase (FAK) is activated in many cancers including PDAC and promotes cancer progression and metastasis. Paxillin, an intracellular adaptor protein that plays a key role in cytoskeletal organization, connects integrins to FAK and plays a key role in assembly and disassembly of focal adhesions. Here, we have reviewed evidence in support of FAK as a potential therapeutic target and summarized related combinatorial therapies., Competing Interests: None.

Epidemiologic data show the incidence of gastric cancer in men is twofold higher than in women worldwide. Oestrogen is reported to have the capacity against gastric cancer development. Endogenous oestrogen reduces gastric cancer incidence in women. Cancer patients treated with oestrogens have a lower subsequent risk of gastric cancer. Accumulating studies report that bone marrow mesenchymal stem cells (BMMSCs) might contribute to the progression of gastric cancer through paracrine effect of soluble factors. Here, we further explore the effect of oestrogen on BMMSCs-mediated human gastric cancer invasive motility. We founded that HBMMSCs notably secrete interleukin-8 (IL-8) protein. Administration of IL-8 specific neutralizing antibody significantly inhibits HBMMSCs-mediated gastric cancer motility. Treatment of recombinant IL-8 soluble protein confirmed the role of IL-8 in mediating HBMMSCs-up-regulated cell motility. IL-8 up-regulates motility activity through Src signalling pathway in human gastric cancer. We further observed that 17β -estradiol inhibit HBMMSCS-induced cell motility via suppressing activation of IL8-Src signalling in human gastric cancer cells. 17β-estradiol inhibits IL8-up-regulated Src downstream target proteins including p-Cas, p-paxillin, p-ERK1/2, p-JNK1/2, MMP9, tPA and uPA. These results suggest that 17β-estradiol significantly inhibits HBMMSCS-induced invasive motility through suppressing IL8-Src signalling axis in human gastric cancer cells., (© 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Metastasis is a complex process that is regulated by multiple signaling pathways, with the focal adhesion kinase (FAK)/paxillin pathway playing a major role in the formation of focal adhesions and cell motility. N-myc downstream regulated gene-1 (NDRG1) is a potent metastasis suppressor in many solid tumor types, including prostate and colon cancer. Considering the antimetastatic effect of NDRG1 and the crucial involvement of the FAK/paxillin pathway in cellular migration and cell-matrix adhesion, we assessed the effects of NDRG1 on this important oncogenic pathway. In the present study, NDRG1 overexpression and silencing models of HT29 colon cancer and DU145 prostate cancer cells were used to examine the activation of FAK/paxillin signaling and the formation of focal adhesions. The expression of NDRG1 resulted in a marked and significant decrease in the activating phosphorylation of FAK and paxillin, whereas silencing of NDRG1 resulted in an opposite effect. The expression of NDRG1 also inhibited the formation of focal adhesions as well as cell migration and cell-collagen adhesion. Incubation of cells with novel thiosemicarbazones, namely di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone and di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone, that upregulate NDRG1 also resulted in decreased phosphorylation of FAK and paxillin. The ability of these thiosemicarbazones to inhibit cell migration and metastasis could be mediated, at least in part, through the FAK/paxillin pathway., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

The parasite, Cryptosporidium parvum, induces human gastroenteritis through infection of host epithelial cells in the small intestine. During the initial stage of infection, C. parvum is reported to engage host mechanisms at the host cell-parasite interface to form a parasitophorous vacuole. We determined that upon infection, the larger molecular weight proteins in human small intestinal epithelial host cells (FHs 74 Int) appeared to globally undergo tyrosine dephosphorylation. In parallel, expression of the cytoplasmic protein tyrosine phosphatase Src homology-2 domain-containing phosphatase 2 (SHP-2) increased in a time-dependent manner. SHP-2 co-localized with the C. parvum sporozoite and this interaction increased the rate of C. parvum infectivity through SH2-mediated SHP-2 activity. Furthermore, we show that one potential target that SHP-2 acts upon is the focal adhesion protein, paxillin, which undergoes moderate dephosphorylation following infection, with inhibition of SHP-2 rescuing paxillin phosphorylation. Importantly, treatment with an inhibitor to SHP-2 and with an inhibitor to paxillin and Src family kinases, effectively decreased the multiplicity of C. parvum infection in a dose-dependent manner. Thus, our study reveals an important role for SHP-2 in the pathogenesis of C. parvum. Furthermore, while host proteins can be recruited to participate in the development of the electron dense band at the host cell-parasite interface, our study implies for the first time that SHP-2 appears to be recruited by the C. parvum sporozoite to regulate infectivity. Taken together, these findings suggest that SHP-2 and its down-stream target paxillin could serve as targets for intervention.

We have established that HCMV acts as a specific ligand engaging and activating cellular integrins on monocytes. As a result, integrin signaling via Src activation leads to the functional activation of paxillin required for efficient viral entry and for the biological changes in monocytes needed for viral dissemination. These biological/molecular changes allow HCMV to use monocytes as "vehicles" for systemic spread and the establishment of lifelong persistence. However, it remains unresolved how HCMV specifically induces this observed monocyte activation. It was previously demonstrated that the HCMV gH/gL/UL128-131 glycoprotein complex facilitates viral entry into biologically relevant cell types. Nevertheless, the mechanism by which the gH/gL/UL128-131 complex promotes this process is unknown. We now show that only HCMV virions possessing the gH/gL/UL128-131 complex are capable of activating integrin/Src/paxillin-signaling in monocytes. In fibroblasts, this signaling is reversed, such that virus lacking the gH/gL/UL128-131 complex is the only virus able to induce the paxillin activation cascade. The presence of the gH/gL/UL128-131 complex also may have an inhibitory effect on integrin-mediated signaling pathway in fibroblasts. Furthermore, we demonstrate that the presence of the gH/gL/UL128-131 complex on the viral envelope, through its activation of the integrin/Src/paxillin pathway, is necessary for efficient HCMV internalization into monocytes and that appropriate actin and dynamin regulation is critical for this entry process. Importantly, productive infection in monocyte-derived macrophages was seen only in cells exposed to HCMV expressing the gH/gL/UL128-131 complex. From our data, the HCMV gH/gL/U128-131 complex emerges as the specific ligand driving the activation of the receptor-mediated signaling required for the regulation of the actin cytoskeleton and, consequently, for efficient and productive internalization of HCMV into monocytes. To our knowledge, our studies demonstrate a possible molecular mechanism for why the gH/gL/UL128-131 complex dictates HCMV tropism and why the complex is lost as clinical isolates are passaged in the laboratory.

Increased sialyltransferase (ST) activity promotes cancer cell metastasis, and overexpression of cell surface sialic acid correlates with poor prognosis in cancer patients. To seek therapies targeting metastasis for cancer treatment, we developed a novel ST inhibitor, Lith-O-Asp, and investigated its antimetastatic and antiangiogenic effects and mechanisms. We found that cells treated with Lith-O-Asp showed a reduction of activity on various ST enzymes by in vitro and cell-based activity analyses. Lith-O-Asp inhibited migration and invasion abilities in various cancer cell lines and showed inhibitory effect on the angiogenic activity of human umbilical vein endothelial cells. Indeed, Lith-O-Asp treatment consequently delayed cancer cell metastasis in experimental and spontaneous metastasis assays in animal models. Importantly, Lith-O-Asp decreased the sialic acid modification of integrin-β1 and inhibited the expression of phospho-FAK, phospho-paxillin, and the matrix metalloprotease (MMP) 2 and MMP9. Lith-O-Asp attenuated the Rho GTPase activity leading to actin dynamic impairment. In addition, 2DE-MS/MS and immunoblotting analyses showed that Lith-O-Asp altered the protein expression level and phosphorylation status of various proteins involved in crucial metastasis and angiogenesis pathways such as vimentin and ribonuclease/angiogenin inhibitor RNH1. Furthermore, Lith-O-Asp treatment significantly inhibited the invasive ability exerted by ectopic overexpression of various ST enzymes catalyzing α-2,6- or α-2,3-sialylation. Our results provide compelling evidence that the potential pan-ST inhibitor, Lith-O-Asp, suppressed cancer cell metastasis likely by inhibiting FAK/paxillin signaling and expressing antiangiogenesis factors. Lith-O-Asp is worthy for further testing as a novel antimetastasis drug for cancer treatment., (© 2011 AACR.)

Purpose: To determine the efficacy of AZD0530, an orally active small molecule Src inhibitor, in human pancreatic cancer xenografts and to seek biomarkers predictive of activity., Experimental Design: Sixteen patient-derived pancreatic cancer xenografts from the PancXenoBank collection at Johns Hopkins were treated with AZD0530 (50 mg/kg/day, p.o.) for 28 days. Baseline gene expression profiles of differently expressed genes in 16 tumors by Affymetrix U133 Plus 2.0 gene array were used to predict AZD0530 sensitivity in an independent group of eight tumors using the K-Top Scoring Pairs (K-TSP) method., Results: Three patient tumors of 16 were found to be sensitive to AZD0530, defined as tumor growth <50% compared with control tumors (100%). Western blot and/or immunohistochemistry results showed that AZD0530 administration resulted in the down-regulation of Src, FAK, p-FAK, p-paxillin, p-STAT-3, and XIAP in sensitive tumor xenografts compared with control tumors. The K-TSP classifier identified one gene pair (LRRC19 and IGFBP2) from the 16 training cases based on a decision rule. The classifier achieved 100% and 83.3% of sensitivity and specificity in an independent test set that consists of eight xenograft cases., Conclusions: AZD0530 treatment significantly inhibits the tumor growth in a subset of human pancreatic tumor xenografts. One gene pair (LRRC19 and IGFBP2) identified by the K-TSP classifier has high predictive power for AZD0530 sensitivity, suggesting the potential for this gene pair as biomarker for pancreatic tumor sensitivity to AZD0530.

Physical forces can activate colon cancer cell adhesion, critical for metastasis. Paxillin is phosphorylated by FAK and required for pressure-stimulated adhesion. However, whether paxillin acts as an inert scaffolding protein or whether paxillin phosphorylation is required is unknown. Transfection with paxillin point-phosphorylation mutants demonstrated that phosphorylation at tyrosines 31 and 118 together is necessary for pressure-stimulated adhesion. We further evaluated potential paxillin partners. Reducing the adaptor protein Crk or the focal adhesion protein p130Cas blocked pressure-stimulated adhesion. Furthermore, Crk and p130Cas both displayed increased co-immunoprecipitation with paxillin in response to increased pressure, except in cells transfected with a Y31Y118 paxillin mutant. Inhibiting the small GTPase Rac1 also abolished pressure-stimulated adhesion, and reducing paxillin by siRNA blocked Rac1 phosphorylation by pressure. Thus, paxillin phosphorylation at tyrosines 31 and 118 together is necessary for pressure-induced adhesion. Paxillin, Crk and Cas form a trimeric complex that activates Rac1 and mediates this effect.

Antagonists to alpha4 integrin show promise for several autoimmune and inflammatory diseases but may exhibit mechanism-based toxicities. We tested the capacity of blockade of alpha4 integrin signaling to perturb functions involved in inflammation, while limiting potential adverse effects. We generated and characterized mice bearing a Y991A mutation in alpha4 integrin [alpha4(Y991A) mice], which blocks paxillin binding and inhibits alpha4 integrin signals that support leukocyte migration. In contrast to the embryonic-lethal phenotype of alpha4 integrin-null mice, mice bearing the alpha4(Y991A) mutation were viable and fertile; however, they exhibited defective recruitment of mononuclear leukocytes into thioglycollate-induced peritonitis. Alpha4 integrins are essential for definitive hematopoiesis; however, the alpha4(Y991A) mice had intact lymphohematopoiesis and, with the exception of reduced Peyer's patches, normal architecture and cellularity of secondary lymphoid tissues. We conclude that interference with alpha4 integrin signaling can selectively impair mononuclear leukocyte recruitment to sites of inflammation while sparing vital functions of alpha4 integrins in development and hematopoiesis.

Histidine-rich glycoprotein (HRGP) is an abundant heparin-binding plasma protein that efficiently arrests growth and vascularization of mouse tumor models. We have shown that the antiangiogenic effect of HRGP is dependent on its histidine/proline-rich domain, which needs to be released from the mother protein to exert its effects. Here we identify a 35-amino-acid peptide, HRGP330, derived from the histidine/proline-rich domain as endowed with antiangiogenic properties in vitro and in vivo. The mechanism of action of HRGP330 involves subversion of focal adhesion function by disruption of integrin-linked kinase (ILK) and focal adhesion kinase (FAK) functions, inhibition of vascular endothelial growth factor (VEGF)-induced tyrosine phosphorylation of the FAK substrate alpha-actinin, and, as a consequence, an arrest in endothelial cell motility. The disturbed focal adhesion function is reflected in the ability of HRGP as well as of HRGP330 to prevent endothelial cell adhesion to vitronectin in a manner involving alpha(v)beta3 integrin. In conclusion, HRGP330, which we define as the minimal antiangiogenic domain of HRGP, exerts its effects through signal transduction targeting focal adhesions, thereby interrupting VEGF-induced endothelial cell motility.