c-Src Signaling Research Articles

Hepatocyte Growth Factor Increases Osteopontin Expression in Human Osteoblasts through PI3K, Akt, c-Src, and AP-1 Signaling Pathway

BackgroundHepatocyte growth factor (HGF) has been demonstrated to stimulate osteoblast proliferation and participated bone remodeling. Osteopontin (OPN) is a secreted phosphoglycoprotein that belongs to the SIBLING family and is present during bone mineralization. However, the effects of HGF on OPN expression in human osteoblasts are large unknown.Methodology/Principal FindingsHere we found that HGF induced OPN expression in human osteoblasts dose-dependently. HGF-mediated OPN production was attenuated by c-Met inhibitor and siRNA. Pretreatment of osteoblasts with PI3K inhibitor (Ly294002), Akt inhibitor, c-Src inhibitor (PP2), or AP-1 inhibitor (curcumin) blocked the potentiating action of HGF. Stimulation of osteoblasts with HGF enhanced PI3K, Akt, and c-Src activation. In addition, incubation of cells with HGF also increased c-Jun phosphorylation, AP-1-luciferase activity, and c-Jun binding to the AP-1 element on the OPN promoter. HGF-mediated AP-1-luciferase activity and c-Jun binding to the AP-1 element was reduced by c-Met inhibitor, Ly294002, Akt inhibitor, and PP2.Conclusions/SignificanceOur results suggest that the interaction between HGF and c-Met increases OPN expression in human osteoblasts via the PI3K, Akt, c-Src, c-Jun, and AP-1 signaling pathway.

Thrombin induces heme oxygenase-1 expression in human synovial fibroblasts through protease-activated receptor signaling pathways

IntroductionThrombin is a key factor in the stimulation of fibrin deposition, angiogenesis, and proinflammatory processes. Abnormalities in these processes are primary features of osteoarthritis (OA). Heme oxygenase (HO)-1 is a stress-inducible rate-limiting enzyme in heme degradation that confers cytoprotection against oxidative injury. Here, we investigated the intracellular signaling pathways involved in thrombin-induced HO-1 expression in human synovial fibroblasts (SFs).MethodsThrombin-mediated HO-1 expression was assessed with quantitative real-time (q)PCR. The mechanisms of action of thrombin in different signaling pathways were studied by using Western blotting. Knockdown of protease-activated receptor (PAR) proteins was achieved by transfection with siRNA. Chromatin immunoprecipitation assays were used to study in vivo binding of Nrf2 to the HO-1 promoter. Transient transfection was used to examine HO-1 activity.ResultsOsteoarthritis synovial fibroblasts (OASFs) showed significant expression of thrombin, and expression was higher than in normal SFs. OASFs stimulation with thrombin induced concentration- and time-dependent increases in HO-1 expression. Pharmacologic inhibitors or activators and genetic inhibition by siRNA of protease-activated receptors (PARs) revealed that the PAR1 and PAR3 receptors, but not the PAR4 receptor, are involved in thrombin-mediated upregulation of HO-1. Thrombin-mediated HO-1 expression was attenuated by thrombin inhibitor (PPACK), PKCδ inhibitor (rottlerin), or c-Src inhibitor (PP2). Stimulation of cells with thrombin increased PKCδ, c-Src, and Nrf2 activation.ConclusionOur results suggest that the interaction between thrombin and PAR1/PAR3 increases HO-1 expression in human synovial fibroblasts through the PKCδ, c-Src, and Nrf2 signaling pathways.

Gq Protein-induced Apoptosis Is Mediated by AKT Kinase Inhibition That Leads to Protein Kinase C-induced c-Jun N-terminal Kinase Activation

G(q) protein-coupled receptors (G(q)PCRs) regulate various cellular processes, including mainly proliferation and differentiation. In a previous study we found that in prostate cancer cells, the G(q)PCR of gonadotropin-releasing hormone (GnRH) induces apoptosis by reducing the PKC-dependent AKT activity and elevating JNK phosphorylation. Because it was thought that G(q)PCRs mainly induce activation of AKT, we first undertook to examine how general this phenomenon is. In a screen of 21 cell lines we found that PKC activation results in the reduction of AKT activity, which correlates nicely with JNK activation and in some cases with apoptosis. To understand further the signaling pathways involved in this stimulation, we studied in detail SVOG-4O and αT3-1 cells. We found that prostaglandin F2α and GnRH agonist (GnRH-a) indeed induce significant Gα(q)- and PKC-dependent apoptosis in these cells. This is mediated by two signaling branches downstream of PKC, which converge at the level of MLK3 upstream of JNK. One branch consists of c-Src activation of the JNK cascade, and the second involves reduction of AKT activity that alleviates its inhibitory effect on MLK3 to allow the flow of the c-Src signal to JNK. At the MAPKK level, we found that the signal is transmitted by MKK7 and not MKK4. Our results present a general mechanism that mediates a G(q)PCR-induced, death receptor-independent, apoptosis in physiological, as well as cancer-related systems.

Evidence that the kinase-truncated c-Src regulates NF-κB signaling by targeting NEMO.

The tyrosine kinase c-Src and transcription factor NF-κB are considered crucial components required for normal osteoclastogenesis. Genetic ablation of either pathway leads to detrimental osteopetrotic phenotypes in mice. Similarly, obstruction of either pathway halts osteoclastogenesis and lessens various forms of bone loss. It has been shown previously that mice expressing a kinase domain-truncated c-Src, termed Src251, develop severe osteopetrosis owing to increased osteoclast apoptosis. It was further suggested that this phenomenon is associated with reduced Akt kinase activity. However, the precise mechanism underlying the osteoclast inhibitory effect of Src251 remains obscure. C-Src associates with TRAF6-p62 interacting with receptor activator of NF-κB (RANK) distal region and the complex facilitate activation of RANK down stream signal transduction cascades including NF-κB. Given this proximity between c-Src and NF-κB signaling in osteoclasts, we surmised that inhibition of osteoclastogenesis by Src251 may be achieved through inhibition of NF-κB signaling. We have demonstrated recently that NEMO, the regulatory subunit of the IKK complex, is crucial for osteoclastogenesis and interacts with c-Src in osteoclast progenitors. Transfection studies, in which we employed various forms of c-Src and NEMO, revealed that the dominant negative form of c-Src, namely Src251, mediates degradation of NEMO thus halting NF-κB signaling. Furthermore, degradation of NEMO requires its intact zinc finger domain which is located at the ubiquitination domain. This process also requires appropriate cellular localization of Src251, since deletion of its myristoylation domain ablates its degradation capacity. Buttressing these findings, the expression of NEMO and NF-κB signaling were significantly reduced in monocytes collected from Src251 transgenic mice.

Involvement of c-Src/STAT3 signal in EGF-induced proliferation of rat spermatogonial stem cells

Epidermal growth factor (EGF) plays a role in male germ cell development, but the precise function is yet to be defined. This study shows that EGF stimulates rat spermatogonial proliferation in a dose-dependent manner and significantly increased the protein levels of phosphated c-Src (p-c-Src) and phosphated signal transducer and activator of transcription 3 (p-STAT3). Moreover, overexpression of c-Src tagged with enhanced green fluorescence protein (EGFP) in rat spermatogonial stem cells enhances the cell viability. In contrast, knockdown or inhibition of c-Src inhibits rat spermatogonial stem cell proliferation; EGF could not abrogate the inhibitory effect. Evidently, the content of p-STAT3 protein was increased in c-Src-expressing cells and decreased in c-Src-suppressing cells. Furthermore, knockdown or inhibition of STAT3 also suppressed cell viability; neither EGF nor increased c-Src could reverse the inhibitory effect. These results are the first evidence that EGF induces proliferation of rat spermatogonial stem cells through c-Src/STAT3 signal.

Genistein Stimulates MCF-7 Breast Cancer Cell Growth by Inducing Acid Ceramidase (ASAH1) Gene Expression

Sphingolipid metabolites, such as ceramide (Cer), sphingosine (SPH), and sphingosine 1-phosphate (S1P), contribute to multiple aspects of carcinogenesis including cell proliferation, migration, angiogenesis, and tumor resistance. The cellular balance between Cer and S1P levels, for example, is an important determinant of cell fate, with the former inducing apoptosis and the later mitogenesis. Acid ceramidase (ASAH1) plays a pivotal role in regulating the intracellular concentration of these two metabolites by hydrolyzing Cer into SPH, which is rapidly phosphorylated to form S1P. Genistein is a phytoestrogen isoflavone that exerts agonist and antagonist effects on the proliferation of estrogen-dependent MCF-7 cells in a dose-dependent manner, primarily as a ligand for estrogen receptors. Genistein can also activate signaling through GPR30, a G-protein-coupled cell surface receptor. Based on the relationship between bioactive sphingolipids and tumorigenesis, we sought to determine the effect of genistein on ASAH1 transcription in MCF-7 breast cancer cells. We show herein that nanomolar concentrations of genistein induce ASAH1 transcription through a GPR30-dependent, pertussis toxin-sensitive pathway that requires the activation of c-Src and extracellular signal regulated kinase 1/2 (ERK1/2). Activation of this pathway promotes histone acetylation and recruitment of phospho-estrogen receptor α and specificity protein-1 to the ASAH1 promoter, ultimately culminating in increased ceramidase activity. Finally, we show that genistein stimulates cyclin B2 expression and cell proliferation in an ASAH1-dependent manner. Collectively, these data identify a mechanism through which genistein promotes sphingolipid metabolism and support a role for ASAH1 in breast cancer cell growth.

Pan-Src Family Kinase Inhibitors Replace Sox2 during the Direct Reprogramming of Somatic Cells

Pan-Src Family Kinase Inhibitors Replace Sox2 during the Direct Reprogramming of Somatic Cells

HMGB‐1 induces IL‐6 production in human synovial fibroblasts through c‐Src, Akt and NF‐κB pathways

High mobility group box chromosomal protein 1 (HMGB-1) is a widely studied, ubiquitous nuclear protein that is present in eukaryotic cells, and plays a crucial role in inflammatory response. However, the effects of HMGB-1 on human synovial fibroblasts are largely unknown. In this study, we investigated the intracellular signaling pathway involved in HMGB-1-induced IL-6 production in human synovial fibroblast cells. HMGB-1 caused concentration- and time-dependent increases in IL-6 production. HMGB-1-mediated IL-6 production was attenuated by receptor for advanced glycation end products (RAGE) monoclonal antibody (Ab) or siRNA. Pretreatment with c-Src inhibitor (PP2), Akt inhibitor and NF-κB inhibitor (pyrrolidine dithiocarbamate and L-1-tosylamido-2-phenylenylethyl chloromethyl ketone) also inhibited the potentiating action of HMGB-1. Stimulation of cells with HMGB-1 increased the c-Src and Akt phosphorylation. HMGB-1 increased the accumulation of p-p65 in the nucleus, as well as NF-κB luciferase activity. HMGB-1-mediated increase of NF-κB luciferase activity was inhibited by RAGE Ab, PP2 and Akt inhibitor or RAGE siRNA, or c-Src and Akt mutant. Our results suggest that HMGB-1-increased IL-6 production in human synovial fibroblasts via the RAGE receptor, c-Src, Akt, p65, and NF-κB signaling pathways.

Abstract 1046: Differential interactions between c-Src and c-Met mediate resistance to c-Src inhibitors

Abstract Cancers of the head and neck are difficult to treat because both the therapy and the tumor impact essential functions such as speech and swallowing and they can also significantly alter facial appearance. One promising molecular target for which new agents have been developed is the Src family kinases (SFKs). SFK inhibition in cancer cells leads to an universal abrogation of invasion but a variable and weak effects on apoptosis and proliferation. The pathways downstream of c-Src promoting survival are not well-characterized. Because cancer therapy that both decreases invasion and induces significant apoptosis would be ideal for head and neck cancers, we sought to characterize the mechanisms of resistance to SFK inhibition in Head and Neck squamous cell carcinoma (HNSCC). SFKs were inhibited in a panel of oral cancer cell lines and the effects on subsequent survival and signaling were measured. The interactions between c-Src and c-Met were evaluated using immunoprecitation and their intrinsic kinase activity by in vitro kinase assay. Cytotoxicity was measured using an MTT assay and the Chou-Talalay combination index calculated. The in vivo effects of c-Met and SFK inhibitors were assessed using an orthotopic model of oral cancer. SFK inhibition resulted in c-Met inhibition in cell lines that were sensitive to SFK inhibitors, but not in resistant cell lines. Isolated c-Met act as a c-Src substrate in both sensitive and resistant cells, whereas distinct difference exists in c-Src and c-Met interaction in intact sensitive and resistant cells. The epidermal growth factor receptor contributed to c-Met activation in resistant cells whereas in sensitive cells c-Met acts as a direct downstream target for c-Src. We demonstrated the biological consequences of this mechanism in vitro with synergistic cytotoxicity and enhanced apoptosis when SFK and c-Met inhibitors were combined. Likewise, the combination resulted in decreased tumor size in vivo. In conclusion, we demonstrate that sustained c-Met activation can mediate resistance to SFK inhibition. The differences between c-Src and c-Met signaling in sensitive and resistant cells are not due to intrinsic structural changes in c-Src or c-Met, but rather to distinct interactions within the intact cells. The synergistic cytotoxic effects of SFK and c-Met inhibition may be important for the treatment of head and neck cancers and should be tested in future clinical trials. 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 1046. doi:10.1158/1538-7445.AM2011-1046

Distinct Interactions Between c-Src and c-Met in Mediating Resistance to c-Src Inhibition in Head and Neck Cancer

c-Src inhibition in cancer cells leads to an abrogation of invasion but a variable effect on apoptosis. The pathways downstream of c-Src promoting survival are not well characterized. Because cancer therapy that both decreases invasion and induces significant apoptosis would be ideal, we sought to characterize the mechanisms of resistance to c-Src inhibition. c-Src was inhibited in a panel of oral cancer cell lines and subsequent survival and signaling measured. The interactions between c-Src and c-Met were evaluated using immunoprecitation and an in vitro kinase assay. Cytotoxicity was measured and the Chou-Talalay combination index calculated. An orthotopic model of oral cancer was used to assess the effects of c-Met and c-Src inhibitors. Inhibition of c-Src resulted in c-Met inhibition in sensitive cells lines, but not in resistant cell lines. Isolated c-Met was a c-Src substrate in both sensitive and resistant cells, but there was no interaction of c-Src and c-Met in intact resistant cells. To examine the biological consequences of this mechanism, we demonstrated synergistic cytotoxicity, enhanced apoptosis, and decreased tumor size with the combination of c-Src and c-Met inhibitors. Sustained c-Met activation can mediate resistance to c-Src inhibition. These data suggest that the differences between c-Met and c-Src signaling in sensitive and resistant cells are due to distinct factors promoting or inhibiting interactions, respectively, rather than to intrinsic structural changes in c-Src or c-Met. The synergistic cytotoxic effects of c-Src and c-Met inhibition may be important for the treatment of head and neck cancers.

Regulation of Caveolin-1 Expression and Phosphorylation by VEGF in Ovine Amnion Cells

Vascular endothelial growth factor (VEGF) has been implicated in the regulation of vesicular transport of amniotic fluid via caveolae across the amnion. This study tested the hypothesis that VEGF regulates caveolar function by stimulating caveolin-1 expression and phosphorylation in ovine amniotic epithelial cells (oAECs). Using primary cultures of oAECs, caveolin-1 was identified by immunofluorescent staining. Caveolin-1 messenger RNA (mRNA) abundance was determined by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and protein by Western blotting. The effects of VEGF( 165) on caveolin-1 expression and phosphorylation were determined. Caveolin-1 immunoreactivity was detected in oAECs. In response to 10 ng/mL VEGF( 165), caveolin-1 mRNA levels increased whereas the protein levels were unaffected. Furthermore, VEGF stimulated caveolin-1 phosphorylation, an effect abrogated by the inhibition of c-Src protein kinase. These data suggest that VEGF upregulates caveolin-1 activity through c-Src signaling pathways. Our observations support the hypothesis that VEGF regulates amniotic fluid transport across the amnion by stimulating caveolin-1 activity to mediate caveolar function in amnion cells.

Minireview: Steroid Receptor Coactivator-3: A Multifarious Coregulator in Mammary Gland Metastasis

A member of the steroid receptor coactivator (SRC)/p160 family, SRC-3 acts as a coregulator for nuclear receptor (NR) and non-NR transcription factors. Such coregulator pleiotropy enables SRC-3 to influence a myriad of signaling networks that are essential for normal physiology and pathophysiology. Although SRC-3's proliferative role in primary tumor formation in the mammary gland is well established, a role for this oncogenic coregulator in tumor cell motility and invasion has only recently been elucidated. In the nucleus, SRC-3 is required for the execution of the epithelial-mesenchymal transition, a programming step which endows an immotile cancer cell with motile and invasive characteristics. Nuclear SRC-3 is also essential for proteolytic breakdown of the extracellular matrix by matrix-metalloproteinases, a process which enables primary tumor cell invasion into the surrounding stroma. At the plasma membrane, however, a truncated isoform of SRC-3 (SRC-3Δ4) serves as a signaling adaptor for the epidermal growth factor→focal adhesion kinase→c-Src signal transduction pathway, a signaling cascade that is central to growth factor-induced cell migration and invasion. Together, these studies underscore a pivotal role for SRC-3 not only as a proto-oncogene but also as a prometastatic factor during the early steps in the invasion-metastasis cascade. Beyond furnishing critical mechanistic insights into SRC-3's involvement in mammary tumor progression, these findings provide opportunities to develop new approaches for breast cancer diagnosis and intervention.

Prostaglandin E2/EP1 Signaling Pathway Enhances Intercellular Adhesion Molecule 1 (ICAM-1) Expression and Cell Motility in Oral Cancer Cells

Oral squamous cell carcinoma has a striking tendency to migrate and metastasize. Cyclooxygenase (COX)-2, the inducible isoform of prostaglandin (PG) synthase, has been implicated in tumor metastasis. However, the effects of COX-2 on human oral cancer cells are largely unknown. We found that overexpression of COX-2 or exogenous PGE(2) increased migration and intercellular adhesion molecule 1 (ICAM)-1 expression in human oral cancer cells. Using pharmacological inhibitors, activators, and genetic inhibition of EP receptors, we discovered that the EP1 receptor, but not other PGE receptors, is involved in PGE(2)-mediated cell migration and ICAM-1 expression. PGE(2)-mediated migration and ICAM-1 up-regulation were attenuated by inhibitors of protein kinase C (PKC)δ, and c-Src. Activation of the PKCδ, c-Src, and AP-1 signaling pathway occurred after PGE(2) treatment. PGE(2)-induced expression of ICAM-1 and migration activity were inhibited by a specific inhibitor, siRNA, and mutants of PKCδ, c-Src, and AP-1. In addition, migration-prone sublines demonstrated that cells with increased migration ability had higher expression of COX-2 and ICAM-1. Taken together, these results indicate that the PGE(2) and EP1 interaction enhanced migration of oral cancer cells through an increase in ICAM-1 production.

C-Src-mediated Phosphorylation of Thyroid Hormone Receptor-interacting Protein 6 (TRIP6) Promotes Osteoclast Sealing Zone Formation

Osteoclasts resorb bone through the formation of a unique attachment structure called the sealing zone. In this study, a role for thyroid hormone receptor-interacting protein 6 (TRIP6) in sealing zone formation and osteoclast activity was examined. TRIP6 was shown to reside in the sealing zone through its association with tropomyosin 4, an actin-binding protein that regulates sealing dimensions and bone resorptive capacity. Suppression of TRIP6 in mature osteoclasts by RNA interference altered sealing zone dimensions and inhibited bone resorption, whereas overexpression of TRIP6 increased the sealing zone perimeter and enhanced bone resorption. Treatment of osteoclasts with lysophosphatidic acid (LPA), which phosphorylates TRIP6 at tyrosine 55 through a c-Src-dependent mechanism, caused increased association of TRIP6 with the sealing zone, as did overexpression of a TRIP6 cDNA bearing a phosphomimetic mutation at tyrosine 55. Further, LPA treatment caused increases in osteoclast fusion, sealing zone perimeter, and bone resorptive capacity. In contrast, overexpression of TRIP6 containing a nonphosphorylatable amino acid residue at position 55 severely diminished sealing zone formation and bone resorption and suppressed the effects of LPA on the cytoskeleton. LPA effects were mediated through its receptor isoform LPA(2), as indicated by treatments with receptor-specific agonists and antagonists. Thus, these studies suggest that TRIP6 is a critical downstream regulator of c-Src signaling and that its phosphorylation is permissive for its presence in the sealing zone where it plays a positive role in osteoclast bone resorptive capacity.

Abstract 5406: ErbB inhibitory protein [EBIP], a modified ectodomain of EGFR, synergizes with dasatinib to inhibit growth of breast cancer cells

Abstract It is becoming increasingly evident that many solid tumors including breast cancer show increased activation of several growth factor receptors, specifically EGFR and its family members (EGFRs) as well as c-Src, a non-receptor tyrosine kinase that promote proliferation, inhibit apoptosis and induce metastasis. We hypothesize that inhibition of c-Src and EGFRs will be an effective therapeutic strategy for breast cancer. To test our hypothesis we used dasatinib (BMS-354825; Bristol-Myers Squibb), a newly developed highly potent, ATP-competitive Src and Abl kinase inhibitor and ErbB Inhibitory Protein (EBIP) that we generated and found to be an effective pan-ErbB inhibitor. EBIP is composed of 1-448 amino acids of the ectodomain of human EGFR (hEGFR), and the 30 amino acid epitope (known as “U” region) of rat EGFR-Related Protein (ERRP) fused at the carboxy-terminal end. Four different breast cancer cell lines (MDA-MB-468, SKBr-3, MDA-MB-453 and MDA-MB-231) expressing different levels of EGFR and/or its family members were used. Indeed, the combination of dasatinib and EBIP was more effective in inhibiting growth of these breast cancer cell lines than either agent alone. To evaluate further the efficacy of combination therapy we chose MDA-MB-468 cells, which represent triple negative breast cancer. Triple negative breast cancer is characterized by absence of expression of hormone receptors (estrogen receptor; ER and progesterone receptor; PR) and no amplification of HER2. This kind of cancer comprising 15% of all breast cancers, is more frequent among African-American and BRCA1-mutation carriers and is associated with aggressive histology and poor prognosis. In EGFR overexpressing MDA-MB-468 cells, the combination therapy caused a greater induction of apoptosis by activating caspases −9 and 8 and attenuating EGFR and c-Src signaling. The combination therapy was greatly effective in inhibiting aggressive behavior of MDA-MB-468 as evidenced by (a) decreased extracellular invasion (b) colony formation and (c) tubule formation by endothelial cells. Furthermore, the combination therapy was highly effective in inhibiting the growth of xenografts of MDA-MB-468 cells in SCID mice, which could be attributed to inhibition of proliferation and induction of apoptosis, accompanied by decreased activation of EGFR and c-Src. In conclusion, our data suggest that combination treatment of dasatinib and EBIP that target EGFRs and Src could be a highly effective therapeutic strategy for breast cancer, with particular reference to triple negative breast cancer. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5406.

Abstract 488: cSrc is a radioresistance molecule in cancer radiotherapy: cSrc-siRNA transfection augments tumor cell radiosensitivity in vitro and chemical inhibition of Src kinases increases tumor response to radiation

Abstract Background: cSrc is often over-expressed in cancer cells including head and neck squamous cell carcinoma (HNSCC) and is implicated in promoting cell survival, proliferation, angiogenesis, cell adhesion and motility. Its role in tumor resistance to cytotoxic agents, such as radiation, is less clear. Present study investigated (1) the relationship between the level of activated cSrc expression and the degree of cell radioresistance and (2) the role of cSrc in the cellular response to radiation, using both in vitro and in vivo models. Materials and methods: Ten HNSCC lines (FaDu, HN-5, UMSCC-1, HN-30, MDA1386, UMSCC-17B, UMSCC-47, Sqccy-1, SN2 and PCI15B) were assessed for their sensitivity to ionizing radiation by clonogenic survival and for expression levels of active Src kinases using phospho-kinase antibody array kit (Proteome ProfilerTM, R&D Systems, Inc., Minneapolis, MN). cSrc expression levels were validated by Western blot. cSrc-siRNA was transfected by electroporation to suppress cSrc expression, after which the cells were assessed for radiosensitivity. We also tested the effect of dasatinib, an inhibitor of Src kinases, on in vitro cell radiosensitivity, and on the growth of tumor xenografts in nude mice. Results: HNSCC lines exhibited a wide range of both radiosensitivity and levels of phosphorylated Src family kinases. There was a highly positive correlation (r=0.883) between SF2 values (cell survival at 2 Gy) and the degree of basal phosphorylated cSrc kinase expression. As the latter increased the ability of cells to survive radiation, increased. Inhibition of cSrc expression by cSrc-siRNA in 2 cell lines tested (FaDu and UMSCC1) resulted in a significant increase in their radiosensitivity, suggesting a causal relationship between cSrc and cell radioresistance. Inhibition of cSrc by dasatinib also resulted in increase of radiosensitivity of 3 cell lines tested (FaDu, HN-5, UMSCC-1). Additionally, dasatinib enhanced in vivo tumor response to radiation as shown by a marked increase in radiation-induced growth delay of FaDu xenografts. Conclusions: Our data showed that (1) a strong correlation exists between the expression level of cSrc and cell radiosensitivity of HNSCCs, (2) inhibition of cSrc expression by siRNA can render tumor cells more radiosensitive, implicating a causal relationship between cSrc and radioresistance, and (3) dasatinib enhanced both the in vitro tumor cell radiosensitivity and in vivo tumor response to radiation. These results suggest that targeting cSrc signaling network may have potential to improve the efficacy of radiotherapy. Supported in part by Bristol-Myers Squibb Company Sponsored Agreement (PI: U. Raju), NIH PO-1 Grant CA-06294 (PI: KK. Ang) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 488.

Histone Deacetylase-3 Activation Promotes Tumor Necrosis Factor-α (TNF-α) Expression in Cardiomyocytes during Lipopolysaccharide Stimulation

Lipopolysaccharides (LPS) induce tumor necrosis factor-alpha (TNF-alpha) production in cardiomyocytes, which contributes to myocardial depression during sepsis. However, the underlying mechanisms remain not fully understood. This study was undertaken to investigate the contribution of histone deacetylase (HDAC) to TNF-alpha expression in cardiomyocytes and the signaling mechanism of LPS-induced HDAC activation. Here, we show for the first time that LPS increases HDAC activity and that inhibition of HDAC decreases LPS-stimulated TNF-alpha expression via the accumulation of NF-kappaB/p65 at the TNF-alpha promoter in cardiomyocytes. Using a positive screen, we have further identified HDAC3 as a specific member of the HDAC family able to regulate TNF-alpha production. Furthermore, our data reveal that LPS-induced HDAC activity is mediated through reactive oxygen species from mitochondria and c-Src signaling. In summary, this study demonstrates a novel signaling mechanism by which LPS via mitochondrial reactive oxygen species/c-Src/HDAC3 pathways mediate TNF-alpha expression in cardiomyocytes.

Cyclooxygenase-2 enhances α2β1 integrin expression and cell migration via EP1 dependent signaling pathway in human chondrosarcoma cells

BackgroundCyclooxygenase (COX)-2, the inducible isoform of prostaglandin (PG) synthase, has been implicated in tumor metastasis. Interaction of COX-2 with its specific EP receptors on the surface of cancer cells has been reported to induce cancer invasion. However, the effects of COX-2 on migration activity in human chondrosarcoma cells are mostly unknown. In this study, we examined whether COX-2 and EP interaction are involved in metastasis of human chondrosarcoma.ResultsWe found that over-expression of COX-2 or exogenous PGE2 increased the migration of human chondrosarcoma cells. We also found that human chondrosarcoma tissues and chondrosarcoma cell lines had significant expression of the COX-2 which was higher than that in normal cartilage. By using pharmacological inhibitors or activators or genetic inhibition by the EP receptors, we discovered that the EP1 receptor but not other PGE receptors is involved in PGE2-mediated cell migration and α2β1 integrin expression. Furthermore, we found that human chondrosarcoma tissues expressed a higher level of EP1 receptor than normal cartilage. PGE2-mediated migration and integrin up-regulation were attenuated by phospholipase C (PLC), protein kinase C (PKC) and c-Src inhibitor. Activation of the PLCβ, PKCα, c-Src and NF-κB signaling pathway after PGE2 treatment was demonstrated, and PGE2-induced expression of integrin and migration activity were inhibited by the specific inhibitor, siRNA and mutants of PLC, PKC, c-Src and NF-κB cascades.ConclusionsOur results indicated that PGE2 enhances the migration of chondrosarcoma cells by increasing α2β1 integrin expression through the EP1/PLC/PKCα/c-Src/NF-κB signal transduction pathway.

Lipoteichoic acid enhances IL-6 production in human synovial fibroblasts via TLR2 receptor, PKCδ and c-Src dependent pathways

Patients with rheumatoid arthritis (RA) are at increased risk of developing infections and appear to be particularly susceptible to septic arthritis. Lipoteichoic acid (LTA), a cell wall component of Gram-positive bacteria is an amphiphilic, negatively charged glycolipid. However, the effects of LTA on human synovial fibroblasts are largely unknown. We investigated the signaling pathway involved in IL-6 production stimulated by LTA in rheumatoid arthritis synovial fibroblasts (RASF). LTA caused concentration- and time-dependent increases in IL-6 production. LTA-mediated IL-6 production was attenuated by Toll-like receptor 2 (TLR2) monoclonal antibody or siRNA. Pretreatment with PKCδ inhibitor (rottlerin), c-Src inhibitor (PP2), AP-1 inhibitor (tanshinone IIA) and NF-κB inhibitor (PDTC and TPCK) also inhibited the potentiating action of LTA. However, focal adhesion kinase (FAK) mutant and siRNA did not affect LTA-mediated IL-6 production. Stimulation of cells with LTA increased the PKCδ and c-Src phosphorylation and kinase activity. LTA increased the accumulation of p-c-Jun and p-p65 in the nucleus, as well as AP-1 and NF-κB luciferase activity. LTA-mediated increase of AP-1 and NF-κB luciferase activity was inhibited by rottlerin and PP2 or TLR2 and PKCδ siRNA or c-Src mutant. Our results suggest that LTA-increased IL-6 production in human synovial fibroblasts via the TLR2 receptor, PKCδ, c-Src, AP-1 and NF-κB signaling pathways.

Glucosylceramide synthase upregulates MDR1 expression in the regulation of cancer drug resistance through cSrc and β-catenin signaling

BackgroundDrug resistance is the outcome of multiple-gene interactions in cancer cells under stress of anticancer agents. MDR1 overexpression is most commonly detected in drug-resistant cancers and accompanied with other gene alterations including enhanced glucosylceramide synthase (GCS). MDR1 encodes for P-glycoprotein that extrudes anticancer drugs. Polymorphisms of MDR1 disrupt the effects of P-glycoprotein antagonists and limit the success of drug resistance reversal in clinical trials. GCS converts ceramide to glucosylceramide, reducing the impact of ceramide-induced apoptosis and increasing glycosphingolipid (GSL) synthesis. Understanding the molecular mechanisms underlying MDR1 overexpression and how it interacts with GCS may find effective approaches to reverse drug resistance.ResultsMDR1 and GCS were coincidently overexpressed in drug-resistant breast, ovary, cervical and colon cancer cells; silencing GCS using a novel mixed-backbone oligonucleotide (MBO-asGCS) sensitized these four drug-resistant cell lines to doxorubicin. This sensitization was correlated with the decreased MDR1 expression and the increased doxorubicin accumulation. Doxorubicin treatment induced GCS and MDR1 expression in tumors, but MBO-asGCS treatment eliminated "in-vivo" growth of drug-resistant tumor (NCI/ADR-RES). MBO-asGCS suppressed the expression of MDR1 with GCS and sensitized NCI/ADR-RES tumor to doxorubicin. The expression of P-glycoprotein and the function of its drug efflux of tumors were decreased by 4 and 8 times after MBO-asGCS treatment, even though this treatment did not have a significant effect on P-glycoprotein in normal small intestine. GCS transient transfection induced MDR1 overexpression and increased P-glycoprotein efflux in dose-dependent fashion in OVCAR-8 cancer cells. GSL profiling, silencing of globotriaosylceramide synthase and assessment of signaling pathway indicated that GCS transfection significantly increased globo series GSLs (globotriaosylceramide Gb3, globotetraosylceramide Gb4) on GSL-enriched microdomain (GEM), activated cSrc kinase, decreased β-catenin phosphorylation, and increased nuclear β-catenin. These consequently increased MDR1 promoter activation and its expression. Conversely, MBO-asGCS treatments decreased globo series GSLs (Gb3, Gb4), cSrc kinase and nuclear β-catenin, and suppressed MDR-1 expression in dose-dependent pattern.ConclusionThis study demonstrates, for the first time, that GCS upregulates MDR1 expression modulating drug resistance of cancer. GSLs, in particular globo series GSLs mediate gene expression of MDR1 through cSrc and β-catenin signaling pathway.