Serum-free Conditioned Medium Research Articles

Identification of CD90 as a marker for lung cancer stem cells in A549 and H446 cell lines

Accumulating evidence supports that cancer stem cells (CSCs) are responsible for tumor initiation, progression, distal metastasis and even drug resistance. Although CD90 has been identified as a marker for several types of stem cells, such as liver CSCs, the potential role of CD90 as a marker for lung CSCs has yet to be fully characterized. Our previous study demonstrated that the lung cancer stem-like cells isolated from A549 tumor spheres, which were cultured in serum-free conditioned medium, had stronger proliferation and self-renewal abilities, and expressed higher levels of the stem cell markers Sox2 and Oct4 as compared to A549 adherent cells. In the present study, we identified CD90 as a novel surface marker of CSCs in lung cancer cells. Furthermore, we isolated CD90+ CSCs from lung cancer cell lines A549 and H446. Our results revealed that the CD90+ cells, but not the CD90- cells, from lung cancer cells displayed higher tumorigenic capacity. These findings suggest that CD90 could be a potential marker of lung CSCs and thus provide new insight into further therapeutic strategies of lung cancer.

Effects of human epidermal growth factor gene‐transfected mesenchymal stem cells on fibroblast migration and proliferation

We were interested in determining whether epidermal growth factor gene-transfected mesenchymal stem cells (EGF-MSC) would accelerate fibroblast migration and proliferation. Fibroblasts were cultured in serum-free conditioned media from EGF-MSC; RT-PCR was performed to detect expression of EGF gene in EGF-MSCs. EGF protein levels in cell culture supernatants from EGF-MSC were assayed by ELISA and proliferation of EGF-MSC-treated fibroblasts was performed using MTT assay. Effects of EGF-MSC on fibroblast migration were evaluated using scratch wound and transmigration assays. Cell adhesion molecules, cell dynamics molecules and phospho-(Ser) kinase substrate expressions of EGF-MSC-treated fibroblasts were evaluated by western blotting. EGF gene expression increased in EGF-MSCs and viability of EGF-MSC-treated fibroblasts was elevated. EGF-MSC-treated fibroblasts showed increased migration compared to controls. Expressions of cell adhesion molecules (β-catenin, N-cadherin), cell dynamics molecules (cofilin, ezrin) and phospho-(Ser) kinase substrates (phospho-MAPK/CDK substrate, phospho-Arg-(Ser)-X-Tyr/Phe-X-pSer motif) increased in EGF-MSC-treated fibroblasts. These results imply that EGF-MSCs contributed to enhancing the wound healing process by increased cell adhesion, dynamic effects, fibroblast migration, and proliferation. This study indicates that EGF-MSCs had a positive influence on fibroblast migration and proliferation and EGF-MSC may provide a useful strategy for wound healing.

Secretory/releasing proteome-based identification of plasma biomarkers in HBV-associated hepatocellular carcinoma

For successful therapy, hepatocellular carcinoma (HCC) must be detected at an early stage. Herein, we used a proteomic approach to analyze the secretory/releasing proteome of HCC tissues to identify plasma biomarkers. Serum-free conditioned media (CM) were collected from primary cultures of cancerous tissues and surrounding noncancerous tissues. Proteomic analysis of the CM proteins permitted the identification of 1365 proteins. The enriched molecular functions and biological processes of the CM proteins, such as hydrolase activity and catabolic processes, were consistent with the liver being the most important metabolic organ. Moreover, 19% of the proteins were characterized as extracellular or membrane-bound. For validation, secretory proteins involved in transforming growth factor-β signaling pathways were validated in plasma samples. Alphafetoprotein (AFP), metalloproteinase (MMP)1, osteopontin (OPN), and pregnancy-specific beta-1-glycoprotein (PSG)9 were significantly increased in HCC patients. The overall performance of MMP1 and OPN in the diagnosis of HCC remained greater than that of AFP. In addition, this study represents the first report of MMP1 as a biomarker with a higher sensitivity and specificity than AFP. Thus, this study provides a valuable resource of the HCC secretome with the potential to investigate serological biomarkers. MMP1 and OPN could be used as novel biomarkers for the early detection of HCC and to improve the sensitivity of biomarkers compared with AFP.

In-depth Characterization of the Secretome of Colorectal Cancer Metastatic Cells Identifies Key Proteins in Cell Adhesion, Migration, and Invasion

Liver metastasis in colorectal cancer is the major cause of cancer-related deaths. To identify and characterize proteins associated with colon cancer metastasis, we have compared the conditioned serum-free medium of highly metastatic KM12SM colorectal cancer cells with the parental, poorly metastatic KM12C cells using quantitative stable isotope labeling by amino acids in cell culture (SILAC) analyses on a linear ion trap-Orbitrap Velos mass spectrometer. In total, 1337 proteins were simultaneously identified in SILAC forward and reverse experiments. For quantification, 1098 proteins were selected in both experiments, with 155 proteins showing >1.5-fold change. About 52% of these proteins were secreted directly or using alternative secretion pathways. GDF15, S100A8/A9, and SERPINI1 showed capacity to discriminate cancer serum samples from healthy controls using ELISAs. In silico analyses of deregulated proteins in the secretome of metastatic cells showed a major abundance of proteins involved in cell adhesion, migration, and invasion. To characterize the tumorigenic and metastatic properties of some top up- and down-regulated proteins, we used siRNA silencing and antibody blocking. Knockdown expression of NEO1, SERPINI1, and PODXL showed a significant effect on cellular adhesion. Silencing or blocking experiments with SOSTDC1, CTSS, EFNA3, CD137L/TNFSF9, ZG16B, and Midkine caused a significant decrease in migration and invasion of highly metastatic cells. In addition, silencing of SOSTDC1, EFNA3, and CD137L/TNFSF9 reduced liver colonization capacity of KM12SM cells. Finally, the panel of six proteins involved in invasion showed association with poor prognosis and overall survival after dataset analysis of gene alterations. In summary, we have defined a collection of proteins that are relevant for understanding the mechanisms underlying adhesion, migration, invasion, and metastasis in colorectal cancer.

Conditioned serum-free medium from umbilical cord mesenchymal stem cells has anti-photoaging properties

Chronic exposure to solar radiation is the primary cause of photoaging and benign and malignant skin tumors. A conditioned serum-free medium (SFM) was prepared from umbilical cord mesenchymal stem cells (UC-MSCs) and its anti-photoaging effect, following chronic UV irradiation in vitro and in vivo, was evaluated. UC-MSC SFM had a stimulatory effect on human dermal fibroblast proliferation and reduced UVA-induced cell death. In addition, UC-MSC SFM blocked UVA inhibition of superoxide dismutase activity. Topical application of UC-MSC SFM to mouse skin prior to UV irradiation blocked the inhibition of superoxide dismutase and glutathione peroxidase activities, and prevented the upregulation of malonaldehyde. UC-MSC SFM thus protects against photoaging induced by UVA and UVB radiation and is a promising candidate for skin anti-photoaging treatments.

Ameloblasts serum-free conditioned medium: bone morphogenic protein 4-induced odontogenic differentiation of mouse induced pluripotent stem cells

Induced pluripotent stem (iPS) cells possess the ability of self-renewal and can differentiate into cells of the three germ layers, both in vitro and in vivo. Here we report a new method to efficiently induce differentiation of mouse iPS cells into the odontogenic lineage. Using ameloblasts serum-free conditioned medium (ASF-CM), we successfully generated ameloblast-like cells from mouse iPS cells. Importantly, culturing mouse iPS cells in ASF-CM supplemented with BMP4 (ASF-BMP4) promoted odontogenic differentiation, which was evident by the upregulation of ameloblast-specific as well as odontoblast-specific genes. On the other hand, culturing mouse iPS cells in ASF-CM supplemented with noggin (ASF-noggin), an inhibitor of BMP4, abrogated this effect. These results suggest that mouse iPS cells can be induced by ASF-BMP4 to differentiate into ameloblast-like and odontoblast-like cells. The results of our study raise the possibility of using patient-specific iPS cells for tooth regeneration in the future. Copyright © 2016 John Wiley & Sons, Ltd.

In vitro evaluation of sialyl Lewis X relationship with head and neck cancer stem cells.

To evaluate in vitro the potential links between sialyl Lewis X (sLeX) and cancer stem cells (CSC) in head and neck squamous cell carcinoma (HNSCC). HNSCC is an aggressive malignancy with high mortality mainly due to metastasis. CSC have emerged as important players in HNSCC metastasis. sLeX is a tetrasaccharide carbohydrate known to play a key role in metastatic dissemination by promoting binding of the tumor cells to the endothelium. Experimental, in vitro. Laboratory of Head and Neck Cancer Metastasis, University of Michigan. A panel of stage- and anatomic-site specific primary and metastatic HNSCC cell lines was assessed by flow cytometry to quantify sLeX relative expression levels. Serum-free conditioned media from the same HNSCC lines was collected over a time course of 72 hours and assessed by Western blot for secreted sLeX expression. Representative HNSCC cell lines were cultured as floating orospheres (condition that enhance CSC growth) or under normal adherent conditions and characterized by flow cytometry for CSC markers (CD44, aldehyde dehydrogenase [ALDH]) comparatively with sLeX expression. sLeX is predominantly expressed in carcinomas originating from the oral cavity. Secreted sLeX is also found to be high in oral carcinomas and increased over the analyzed time course. Floating orospheres were strongly positive for CD44 and ALDH, confirming CSC enrichment of the orospheres. Tumor cells grown as orospheres are 95% to 100% positive for sLeX compared to 10% to 40% of adherent counterpart. These studies provide the first evidence of sLeX relationship with CSC in HNSCC.

Adipose stromal cells primed with hypoxia and inflammation enhance cardiomyocyte proliferation rate in vitro through STAT3 and Erk1/2

BackgroundExperimental clinical stem cell therapy has been used for more than a decade to alleviate the adverse aftermath of acute myocardial infarction (aMI). The post-infarcted myocardial microenvironment is characterized by cardiomyocyte death, caused by ischemia and inflammation. These conditions may negatively affect administered stem cells. As postnatal cardiomyocytes have a poor proliferation rate, while induction of proliferation seems even more rare. Thus stimulation of their proliferation rate is essential after aMI. In metaplastic disease, the pro-inflammatory cytokine interleukin-6 (IL-6) has been identified as potent mediators of the proliferation rate. We hypothesized that IL-6 could augment the proliferation rate of (slow-)dividing cardiomyocytes.MethodsTo mimic the behavior of therapeutic cells in the post-infarct cardiac microenvironment, human Adipose Derived Stromal Cells (ADSC) were cultured under hypoxic (2% O2) and pro-inflammatory conditions (IL-1β) for 24h. Serum-free conditioned medium from ADSC primed with hypoxia and/or IL-1β was added to rat neonatal cardiomyocytes and adult cardiomyocytes (HL-1) to assess paracrine-driven changes in cardiomyocyte proliferation rate and induction of myogenic signaling pathways.ResultsWe demonstrate that ADSC enhance the proliferation rate of rat neonatal cardiomyocytes and adult HL-1 cardiomyocytes in a paracrine fashion. ADSC under hypoxia and inflammation in vitro had increased the interleukin-6 (IL-6) gene and protein expression. Similar to conditioned medium of ADSC, treatment of rat neonatal cardiomyocytes and HL-1 with recombinant IL-6 alone also stimulated their proliferation rate. This was corroborated by a strong decrease of cardiomyocyte proliferation after addition of IL-6 neutralizing antibody to conditioned medium of ADSC. The stimulatory effect of ADSC conditioned media or IL-6 was accomplished through activation of both Janus Kinase-Signal Transducer and Activator of Transcription (JAK/STAT) and Mitogen-Activated Protein (MAP) kinases (MAPK) mitogenic signaling pathways.ConclusionADSC are promising therapeutic cells for cardiac stem cell therapy. The inflammatory and hypoxic host post-MI microenvironment enhances the regenerative potential of ADSC to promote the proliferation rate of cardiomyocytes. This was achieved in paracrine manner, which warrants the development of ADSC conditioned medium as an “of-the-shelf” product for treatment of post-myocardial infarction complications.

Abstract P6-06-02: Characterization of an exosome-associated apoptosis-inducing activity produced by triple negative breast cancer cells.

Abstract Purpose: We sought to investigate whether breast cancer cells can suppress the proliferation of other breast cancer cells in order to understand the biological significance of cell heterogeneity in breast cancer. Materials and Methods: 20 breast cancer cell lines, corresponding to all molecular profiles of breast cancer, were cultured in a 20×20 conditioned medium protocol. Cell proliferation and apoptosis were evaluated by EdU (5-ethynyl-2′-deoxyuridine) and PARP (Poly ADP ribose) cleaved labeling respectively, and documented by fluorescence microscopy. Serum free conditioned media (CM) were prepared from all the cell lines, 24hrs of incubation, and screened for their anti-proliferative effect against the different breast cancer tumor cells. Active media were ultra-centrifuged and both the supernatants as well as the pellets were assessed for the anti-proliferative activity. Results: Using this 20×20 matrix, CM obtained from two out of six triple negative breast cancer cells displayed anti-proliferative activity against the majority of cell lines, irrespectively of their molecular phenotype including triple negative cells. No significant anti-proliferative activity was observed in CMs from luminal or HER2(+) cell lines. However the two triple negative CMs induced a significant decrease in the cell lines' proliferation index, which was associated with a concomitant increase in cells' apoptotic index. Ultracentrifugation revealed that the apoptosis-inducing activity was retained in the pellet but not the supernatant of the CM. Negative staining electron microscopy (EM) revealed the presence of exosomes in the CM's ultra-centrifuged pellet, which stained positive for cd63 surface protein marker using an immunogold assay. Conclusions: These data strongly suggest that some triple negative breast cancer cell lines posses exosome-mediated apoptosis-inducing activity. This activity may have biological and clinical relevance since it could be a factor participating in the selection of tumor cell subpopulations of different degrees of aggressiveness. Further proteomic and microRNA profiling of the exosomes is ongoing in order to characterize this exosome-mediated apoptosis-inducing activity. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-06-02.

Human Dental Pulp-Derived Stem Cells Protect Against Hypoxic-Ischemic Brain Injury in Neonatal Mice

Perinatal hypoxia-ischemia (HI) has high rates of neurological deficits and mortality. So far, no effective treatment for HI brain injury has been developed. In this study, we investigated the therapeutic effects of stem cells from human exfoliated deciduous teeth (SHED) for the treatment of neonatal HI brain injury. Unilateral HI was induced in postnatal day 5 (P5) mice. Twenty-four hours later, SHED, human skin fibroblasts, or serum-free conditioned medium derived from these cells was injected into the injured brain. The effects of cell transplantation or conditioned medium injection on the animals' neurological and pathophysiological recovery were evaluated. Transplanted SHED, but not fibroblasts, significantly reduced the HI-induced brain-tissue loss and improved neurological function. SHED also improved the survival of the HI mice. The engrafted SHED rarely differentiated into neural lineages; however, their transplantation inhibited the expression of proinflammatory cytokines, increased the expression of anti-inflammatory ones, and significantly reduced apoptosis. Notably, the intracerebral administration of SHED-conditioned medium also significantly improved the neurological outcome, inhibited apoptosis, and reduced tissue loss. SHED transplantation into the HI-injured brain resulted in remarkable neurological and pathophysiological recovery. Our findings indicate that paracrine factors derived from SHED support a neuroprotective microenvironment in the HI brain. SHED graft and SHED-conditioned medium may provide a novel neuroprotective therapy for HI.

Matrix Metalloproteinase-13 (MMP-13) Directly and Indirectly Promotes Tumor Angiogenesis

Matrix metalloproteinases (MMPs) are extracellular zinc-dependent endopeptidases involved in the degradation and remodeling of extracellular matrix in physiological and pathological processes. MMPs also have a role in cell proliferation, migration, differentiation, angiogenesis, and apoptosis. We previously identified cancer invasion-related factors by comparing the gene expression profiles between parent and the highly invasive clone of cancer cells. Matrix metalloproteinase-13 (MMP-13) was identified as a common up-regulated gene by cancer invasion-related factors. Although MMP-13 slightly promoted tumor invasion, we found that MMP-13 was involved in tumor angiogenesis. Conditioned medium from MMP-13-overexpressing cells promoted capillary formation of immortalized human umbilical vein endothelial cells. Furthermore, treatment with recombinant MMP-13 protein enhanced capillary tube formation both in vitro and in vivo. MMP-13-promoted capillary tube formation was mediated by activation of focal adhesion kinase and ERK. Interestingly, MMP-13 promoted the secretion of VEGF-A from fibroblasts and endothelial cells. By immunohistochemical analysis, we found a possible correlation between MMP-13 expression and the number of blood vessels in human cancer cases. In summary, these findings suggest that MMP-13 may directly and indirectly promote tumor angiogenesis.

365 Proteomic Characterization of the Cardiac Fibroblast Exosome

365 Proteomic Characterization of the Cardiac Fibroblast Exosome

SLeX, Biomarker of Metastatic Head and Neck Carcinomas

Objective: Among the clinical challenges of head and neck squamous cell carcinomas (HNSCC), the morbidity and mortality of advanced oral cancer (OC) remains paramount. The molecular mechanism controlling OC progression and metastasis are thus key to the improvement of clinical outcomes. Metastatic dissemination begins with adhesion of circulating tumor cells to the E-selectin-expressing endothelium with high levels of the main E-selectin ligand, sLeX, having been implicated in various carcinomas. Our study represents the first investigation of the sLeX involvement in HNSCC dissemination. Method: A panel of stage- and anatomic site-specific primary and metastatic HNSCC cell lines was examined by flow cytometry to quantify cell-surface sLeX expression. Secreted sLeX expression was measured by Western-blot in serum-free conditioned medium. Quantitative real time PCR (qRT-PCR) was used to identify the fucosyl-transferases involved in sLeX neosynthesis in HNSCC. HNSCC invasive and migratory abilities were evaluated by Matrigel assays according with their sLeX expression levels. Results: HNSCC originating from oral cavity (OC) expressed high sLeX that increased with advanced stage; it was highest in cells from recurrent (HNSCC14B) and metastatic (UMSCC103) tumors with known poor clinical outcomes. The expression of secreted sLeX was also high in OC supernatants. sLeX-positive tumor cells exhibited higher invasive potential than sLex-negative HNSCC. Conclusion: Among various sites HNSCC, both cell-bound and soluble sLeX was predominantly expressed in OC carcinomas and associated with recurrent and metastatic tumors. The mechanism of sLeX involvement in OC progression is under investigation. A better understanding of the HNSCC dissemination will positively impact the clinical management.

Investigation of proNT/NMN secretion from small cell lung carcinoma cells using a mouse xenograft model

Proneurotensin/neuromedin N (proNT/NMN), the precursor of neurotensin (NT) and neuromedin N (NMN), is produced by cancer tissues derived from the pancreas and colon. NT stimulates tumor growth and proliferation through its receptors; however, little is known about the precursor molecule in cancer tissues. We previously demonstrated that proNT/NMN is secreted from small cell lung carcinoma (SCLC) cell lines in serum-free conditioned medium, but not from non-small cell lung carcinoma (NSCLC) cell lines. It was suggested that this precursor may serve as a tumor marker for SCLC. In this study, we established invivo xenograft models to evaluate the possibility of proNT/NMN as a specific tumor marker. SBC3 cells, derived from human SCLC, were inoculated into mice, and the proNT/NMN levels in plasma and tumor tissues were detected using specific antibodies. In contrast to control mouse plasma, the proNT/NMN levels in tumor-bearing mice increased as the tumors grew, and the elevated plasma proNT/NMN levels were decreased by tumor resection. Moreover, proNT/NMN was expressed in SBC3 tumors, suggesting that proNT/NMN was secreted into blood from the tumor, and this secretion may be specific to SCLC.

The ovarian cancer‐derived secretory/releasing proteome: A repertoire of tumor markers

Ovarian cancer is the most lethal gynecological malignancy worldwide, and early detection of this disease using serum or plasma biomarkers may improve its clinical outcome. In the present study, a large scale protein database derived from ovarian cancer was created to enable tumor marker discovery. First, primary organ cultures were established with the tumor tissues and corresponding normal tissues obtained from six ovarian cancer patients, and the serum-free conditioned medium (CM) samples were collected for proteomic analysis. The total proteins from the CM sample were separated by SDS-PAGE, digested with trypsin and then analyzed by LC-MS/MS. Combining data from the tumor tissues and the normal tissues, 1129 proteins were identified in total, of which those categorized as "extracellular proteins" and "plasma membrane proteins" accounted for 21.4% and 16.9%, respectively. For validation, three secretory proteins (NID1, TIMP2, and VCAN) involved in "organ development"-associated subnetwork, showed significant differences between their levels in the circulating plasma samples from ovarian cancer patients and healthy women. In conclusion, this ovarian cancer-derived protein database provides a credible repertoire of potential biomarkers in blood for this malignant disease, and deserves mining further.

All-trans-retinoic acid inhibits growth of head and neck cancer stem cells by suppression of Wnt/β-catenin pathway

Differentiation therapy is a novel approach to eradicate cancer stem cells (CSCs), including head and neck squamous carcinoma CSC (HNSC CSC). All-trans-retinoic acid (ATRA) is a potent differentiating agent. We studied the anti-tumour effect of ATRA on HNSC CSC. HNSC CSCs were differentiated by ATRA in a serum-free conditioned medium. The effect of differentiation on tumour growth was assessed in vitro and in vivo, and chemosensitisation was examined using a colorimetric viability assay. In addition, the involvement of Wnt/β-catenin signalling as an underlying mechanism of the anti-tumour effect of retinoic acid (RA) on HNSC CSCs was assessed. ATRA suppressed the expression of the stem cell markers Oct4, Sox2, Nestin and CD44 in HNSC CSCs and inhibited the proliferation of HNSC CSCs in vitro and in vivo. Furthermore, ATRA treatment augmented the chemosensitising effects of cisplatin. The anti-tumour effects of ATRA may be associated with down-regulation of Wnt/β-catenin signalling. In conclusion, ATRA may be potentially valuable in treatment of HNSC CSC, especially in combination with cisplatin.

Abstract 35: Breast cancer cell interactions

Abstract In this study, a cell culture approach is employed to identify patterns of pair-wise interaction between cancer cells over a panel of 20 established epithelial breast cancer cell lines. It is our hypothesis that different clones within a tumor, as well as different cell lines in cultured conditions, interact in one of two ways: a) by competitive growth or, b) by synergistic growth. We investigate this by culturing cell lines in serum-free conditioned medium (CM), i.e. medium which has already been used on other cell lines. Cell population growth is monitored by incubating cultures with 5-Ethyl-2′-deoxyuridine (EdU). Cell apoptosis is obtained by labeling fixed cultures with a cleaved Poly ADP Ribose Polymerase (c-PARP) monoclonal antibody. Results yield two 20x20 matrices of cell to CM interaction, and confirm that cancer cells interact through secreted substances in the medium. In an effort to trace these phenomenological interactions back to specific biochemical pathways, the interaction matrices are correlated to gene expression data available for these cell lines. Furthermore, the experiment reveals two cell lines, HCC38 and BT20, whose media produce a strong apoptotic and strong anti-proliferative effect respectively. By employing molecular weight fractionation, we attempt to isolate specific components from the media retaining the strong interaction activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 35. doi:1538-7445.AM2012-35

Abstract 5294: Increased angiogenesis resulting from TIMP-1 inhibition of TSP-1 is mediated by PI3kinase and FAK interactions

Abstract Tissue Inhibitor of Matrix Metalloprotease -1 (TIMP-1), one of the four natural inhibitors of matrix metalloproteinases was initially identified for its Erythroid Potentiating Activity. Since then, it has been shown to independently induce cell proliferation and inhibit apoptosis in a number of cell types. TIMP-1 has also been shown to promote, but also more often to inhibit angiogenesis. Previously, we have shown that TIMP-1 overexpression in a lung adenocarcinoma cell line (H2009) resulted in more aggressive and vascular tumors in nude mice and increased capillary network formation and tumorigenicity in vitro. An angiogenesis pathway-specific gene array had identified a 3-fold reduction in thrombospondin 1 (TSP-1) levels in the TIMP-1 overexpressing clone HB-1. TSP-1 siRNA clone exhibited a similar increase in in vitro angiogenesis assays. In the present study, we have sought to identify the signaling pathway involved whereby TIMP-1 inhibits TSP-1. To this end, we have used specific inhibitors to inhibit TIMP1 initiated signaling pathways. MAP kinase, PI3/Akt inhibitors were used, either singly or in combination with Focal Adhesion kinase (FAK) inhibitor. Our results indicate a synergistic interaction between PI3/Akt pathway and FAK signaling, which restores TSP-1 levels by approximately 3-fold relative to control. Gelatin zymography of serum-free conditioned media (SFCM) revealed a reduction in proMMP-9 level in the HB-1 clone versus H2009. Additionally MMP9 specific ELISA reveals a two-fold reduction in MMP9 levels. This is in tune with the role of TSP-1 as an inhibitor of MMP-9 activation such that decreased TSP-1 level results in increased activation of MMP-9 with a concomitant reduction in the extracellular level of proMMP-9. Thus our findings define interaction of TIMP-1 via PI3kinase and FAK signaling. resulting in TSP-1 decrease and subsequent downstream promotion of angiogenesis with increased tumor kinetics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5294. doi:1538-7445.AM2012-5294

Abstract 2427: The stress response controls the interorgan glutathione/interleukin-6 cycling activity: A systemic signaling mechanism promoting metastases growth.

Abstract B16 melanoma F10 (B16-F10) cells with high glutathione (GSH) content show very high metastatic activity in vivo. An intertissue flow of GSH, where the liver is the main reservoir, increases GSH content in metastatic cells and promote their growth. We studied possible tumor-derived molecular signals that could activate GSH release from hepatocytes. GSH efflux increases in hepatocytes isolated from mice bearing liver or lung metastases, thus suggesting a systemic mechanism. Fractionation of serum-free conditioned medium from cultured B16-F10 cells and monoclonal antibody-induced neutralization techniques facilitated identification of interleukin (IL)-6 as a tumor-derived molecule promoting GSH efflux in hepatocytes. IL-6 activates GSH release through a methionine-sensitive/organic anion transporter polypeptide 1- and multidrug resistance protein 1-independent channel located on the sinusoidal site of hepatocytes. Specific siRNAs were used to knock down key factors in the main signaling pathways activated by IL-6, which revealed a STAT3-dependent mechanism. Our results show that IL-6 (mainly of tumor origin) facilitates GSH release from hepatocytes and its interorgan transport to metastatic growing foci. Metastatic tumor activated the hypothalamic-pituitary-adrenal axis, thus increasing expression of hypothalamic corticotropin releasing factor and plasma levels of adrenocorticotropin hormone, corticosterone, and cathecolamines. Corticosterone and norepinephrine increased IL-6 expression and synthesis in metastatic cells, thus promoting the interorgan GSH/IL-6 cycling activity. Our results suggest possible mechanisms of cross-talk between metastases and the central nervous system of the host. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2427. doi:1538-7445.AM2012-2427

Functions of Rhomboid Family Protease RHBDL2 and Thrombomodulin in Wound Healing

The expression of thrombomodulin (TM), a membrane glycoprotein, is upregulated in neoepidermis during cutaneous wound healing. Rhomboid-like-2 (RHBDL2), an intramembrane serine protease, specifically cleaves TM at the transmembrane domain and causes the release of soluble TM (sTM). However, the physiological functions of TM and RHBDL2 in wound healing remain unclear. We demonstrated that both TM and RHBDL2 are upregulated in HaCaT cells stimulated by scratch wounds; furthermore, increased sTM was found in culture medium. Conversely, inhibition of RHBDL2 by 3,4-dichloroisocoumarin (DCI) or short hairpin RNA significantly inhibited wound-induced TM ectodomain shedding and wound healing. Both conditioned media from multiple-scratch-wounded HaCaT and recombinant sTM accelerated wound healing in HaCaT cells; such effects were abrogated by anti-TM antibodies. The RNA released from injured cells is involved in the induction of TM and RHBDL2. RHBDL2 and sTM were upregulated in ex vivo tissue culture of the injured skin. Furthermore, DCI inhibited sTM production and wound healing; this was reversed by recombinant sTM in mice. Thus, RHBDL2 and TM have important roles in wound healing via the release of sTM from keratinocytes; this may function as an autocrine/paracrine signal promoting wound healing.