Akt Signal Transduction Pathway Research Articles

The Inhibitory Effect of 3 β -Hydroxy-12-oleanen-27-oic Acid on Growth and Motility of Human Hepatoma HepG2 Cells through JNK and Akt Signaling Pathway.

3β-Hydroxy-12-oleanen-27-oic acid (ATA) was a main antitumor active triterpene from the rhizomes of Astilbe chinensis. In this study, we investigated its effects on growth, apoptosis, cell cycle, motility/invasion, and metatasis in human hepatoma HepG2 cells in vitro and antimetastasis of B16-F10 melanoma in mice in vivo, as well as its molecular mechanisms of action using a high-throughput Cancer Pathway Finder PCR Array. ATA could not only induce tumor cells into apoptosis through the activation of both extrinsic and intrinsic pathways, arrest HepG2 cells in G2/M phase, but also suppress the invasion and metastasis abilities of HepG2 cells and the lung metastasis of B16-F10 melanoma in mice. PCR array assay revealed that ATA upregulated 9 genes including CDKN1A, MDM2, CFLAR (CASPER), TNFRSF10B (DR5), c-Jun, IL-8, THBS1, SERPINB5 (maspin), and TNF and downregulated 8 genes such as CCNE1, AKT, ANGPT1, TEK, TGFBR1, MMP9, U-PA, and S100A4. These results indicate that ATA could exert antitumor effects through activating JNK/MAPK and suppressing AKT signal transduction pathways and that ATA might be a potent anticancer agent.

Inhibition of PI3K/Akt and Erk Signaling Pathways by BCR-ABL Tyrosine Kinase Inhibitors up-Regulates the Immunosuppressive Receptor Osteoactivin in Monocyte-Derived Dendritic Cells

AbstractAbstract 1050Osteoactivin (GPNMB, DC-HIL) is a type I transmembrane glycoprotein that is expressed in dendritic cells (DC). Osteoactivin/syndecan-4 (SD-4) interaction was previously shown to inhibit T cell activation by antigen-presenting cells. We recently demonstrated that exposure of human peripheral blood monocytes to IL-10 or pharmacological levels of the BCR-ABL tyrosine kinase inhibitors (TKI) imatinib or nilotinib during differentiation into monocyte-derived DC (moDC) causes up-regulation of osteoactivin at the transcript and protein level in vitro (Schwarzbich et al., 2012). Here we aimed to elucidate the molecular mechanisms responsible for osteoactivin up-regulation in moDC upon exposure to IL-10 or TKI (imatinib, nilotinib). moDC were generated from blood monocytes by plastic adherence and exposure to GM-CSF and IL-4. Every second day, starting from the first day of culture, IL-10 or TKI were added to the culture medium. Alternatively, specific inhibitors of PI3K, Akt, Erk and/or C-Raf signal transduction pathways were added to the cell cultures. Cells were harvested on day 7 of culture for immunophenotyping and osteoactivin expression analysis using FACS, western-blotting and real-time qRT-PCR. The expression and activity of signal transduction molecules was monitored by western-blotting and pathway protein arrays. Analysis of moDC function was performed using mixed lymphocyte reactions (MLR). Our in vitro analysis revealed that IL-10 and BCR-ABL TKI inhibit the PI3K/Akt and, to a lesser extent, the Erk pathway in moDC. Specific inhibition of these signal transduction cascades resulted in profound up-regulation of osteoactivin expression in moDC confirming the involvement of PI3K/Akt and Erk in the regulation of osteoactivin expression. In line, TKI treatment and inhibition of PI3K/Akt and Erk comparably affected the phenotype of moDC. Moreover, the inhibition of these signalling cascades resulted in reduced stimulatory capacity of moDC in MLR with allogenic T cells, and this could be restored by addition of blocking osteoactivin antibody. Our data reveal that TKI exert immunosuppressive effects in moDC by interfering with pathways involved in IL-10 receptor signaling. Inhibition of osteoactivin expression or function may thus constitute a promising strategy in combinatory approaches using TKI and DC-based immunotherapy and may enhance the efficacy of immunotherapeutic interventions in cancer patients. Disclosures:No relevant conflicts of interest to declare.

Interleukin‐11 Increases Cell Motility and Up‐Regulates Intercellular Adhesion Molecule‐1 Expression in Human Chondrosarcoma Cells

Interleukin-11 (IL-11) was originally identified as the cytokine that could induce the proliferation of human cells. Recent studies have shown that IL-11 plays a critical role in tumor growth, angiogenesis, and metastasis. Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. However, the effects of IL-11 on human chondrosarcoma cells are largely unknown. Here, we found that IL-11 increased the migration and expression of intercellular adhesion molecule-1 (ICAM)-1 in human chondrosarcoma cells. We also found that human chondrosarcoma tissues had significant expression of the IL-11 which was higher than that in primary chondrocytes. The phosphatidylinositol 3-kinase (PI3K), Akt, and NF-κB pathways were activated by IL-11 treatment, and the IL-11-induced expression of ICAM-1 and migration activity were inhibited by the specific inhibitors and mutant forms of PI3K, Akt, and NF-κB cascades. Taken together, our results indicate that IL-11 enhanced the migration of the chondrosarcoma cells by increasing ICAM-1 expression through the IL-11Rα receptor, PI3K, Akt, and NF-κB signal transduction pathway.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

35 THERAPEUTIC EFFECTS OF INSULIN-LIKE GROWTH FACTOR-1 ON STRESS URINARY INCONTINENCE IN RATS WITH SIMULATED CHILD BIRTH TRAUMA INJURY

You have accessJournal of UrologyUrodynamics/Incontinence/Female Urology: Basic Research I1 Apr 201235 THERAPEUTIC EFFECTS OF INSULIN-LIKE GROWTH FACTOR-1 ON STRESS URINARY INCONTINENCE IN RATS WITH SIMULATED CHILD BIRTH TRAUMA INJURY Yasuhiro Sumino, Satoru Yoshikawa, Hiromitsu Mimata, and Naoki Yoshimura Yasuhiro SuminoYasuhiro Sumino Pittsburgh, PA More articles by this author , Satoru YoshikawaSatoru Yoshikawa Pittsburgh, PA More articles by this author , Hiromitsu MimataHiromitsu Mimata Oita, Japan More articles by this author , and Naoki YoshimuraNaoki Yoshimura Pittsburgh, PA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.078AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Insulin-like growth factor-1 (IGF-1) plays an important role in cell proliferation, survival and regeneration in various tissues. However, the therapeutic potential of IGF-1 for stress urinary incontinence (SUI) has not been explored. We examined the effect of IGF-1 in a rat model of SUI induced by stimulated birth trauma. METHODS Simulated birth trauma was induced by vaginal distension (VD) with an inflated balloon catheter for 4 hrs in the vagina of female SD rats. At 4, 7, 14 and 28 days after VD, (1) functional assessments were performed by measuring leak point pressure (LPP), urethral baseline pressure (UBP) and urethral responses during passive increment in intravesicular pressure and (2) the expression of IGF-1 and IGF-1 receptor (IGF1R) mRNA and protein in damaged tissues was examined by real-time RT-PCR and immunohistochemistry. Thereafter, human recombinant IGF-1 (hrIGF-1) (50 and 150μg/kg/day) or vehicle (saline) was continuously delivered from 1 day before VD using subcutaneous osmotic pumps. At 4 and 7 days after VD, the effect of IGF-1 treatment was examined by ELISA measurement of serum hrIGF-1, functional analyses (LPP, UBP and urethral response), and Western blotting analysis of Akt signal transduction pathways in urethral tissues. RESULTS After 4 and 7 days, VD rats had significantly reduced LPP, UBP and urethral responses, with a recovery to the normal level 14 and 28 days after VD. mRNA and protein levels of IGF-1 and IGF1R were significantly increased in the urethra and pudendal nerves 4 and 7 days after VD. IGF-1 treatment (50 and 150μg/kg/day) increased serum hrIGF-1 levels to 17.8±5.3 and 40.4±12.5 ng/ml, respectively. These IGF-1-treated groups showed significant improvement in LPP, UBP and urethral responses 4 and 7 days after VD (Figure). Moreover, phosphorylated Akt was highly expressed in the IGF-1-treated urethra. CONCLUSIONS IGF-1 treatment accelerates recovery from SUI induced by stimulated child birth trauma in rats, possibly through activation of the Akt signal transduction pathway, suggesting that IGF-1 has an important role in the recovery from childbirth-related SUI. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e14-e15 Peer Review Report Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yasuhiro Sumino Pittsburgh, PA More articles by this author Satoru Yoshikawa Pittsburgh, PA More articles by this author Hiromitsu Mimata Oita, Japan More articles by this author Naoki Yoshimura Pittsburgh, PA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Role of phosphatidylinositol 3-kinase/protein-serine-threonine kinases signal transduction pathway in EphB receptor-mediated neuropathic pain in rats

Objective To investigate the role of phosphatidylinositol 3-kinase (PI3K)/protein-serine-threonine kinases(Akt) signal transduction pathway in EphB receptor-mediated neuropathic pain in rats.Methods Forty-eight pathogen-free male SD rats aged 2-3 months weighing 150-180 g were randomly divided into 6 groups (n =8 each):groups sham operation (groups S1 and S2 ); groups chronic constrictive injury (CCI) (groups C1and C2 ) and groups EphBI-Fc (EphB receptor antagonist) + CCI (groups E1 and E2 ).Neuropathic pain was induced by placing 4 ligatures on left sciatic nerve at 1 mm intervals with 5-0 silk thread in groups C1,C2,E1 and E2.EphBI-Fc 0.5 μg in 5 μl normal saline was injected intrathecally 1 h before operation and at 1 and 2 d after operation (group E1 ) or on 5th day after operation (group E2).Normal saline 5 μl was injected intrathecally instead of EphBI-Fc 1 h before operation and at 1 and 2 days after operation (groups S1 and C1 ) or on 5th day after operation (groups S2 and C2 ).Pain withdrawal latency to noxious thermal stimulation (PWL) and pain withdrawal threshold to noxious mechanical stimulation (PWT) were measured before operation and at 1,3 and 5 d after operation.The animals were sacrificed at 5 d after operation after measurement of pain threshold.The lumbar segment of spinal cord (L4-6) was removed for determination of c-Fos,PI3K and phosphorylated Akt(p-Akt) expression.Results CCI significantly reduced PWL and PWT and up-regulated spinal c-Fos,PI3K and p-Akt expression in groups C1 and C2 as compared with groups S1 and S2.EphB1-Fc significantly decreased hyperalgesia and the upregulated spinal Fos,PI3K and p-Akt protein expression induced by CCI in groups E1 and E2 as compared with groups C1 and C2.Conclusion Spinal EphB receptor is involved in the development and maintenance of neuropathic pain through PI3K/Akt signal transduction pathway. Key words: 1-Phosphatidylinositol 3-kinase; Protein-serine-threonine kinases; Receptors, Eph family; Neuralgia

Jolkinolide B induces apoptosis in MDA-MB-231 cells through inhibition of the PI3K/Akt signaling pathway

The phosphoinositol-3-kinase (PI3K)/Akt signal transduction pathway is critically important for tumor cell growth, proliferation and apoptosis. Apoptosis activation has been reported to be a good target in cancer therapies. In this study, we have found that jolkinolideB (JB), a diterpenoid from the traditional Chinese medicinal herb Euphorbia fischeriana Steud, strongly inhibited the expression of the PI3K p85 subunit and the phosphorylation of Akt. Furthermore, we evaluated the effects of JB on the proliferation and apoptosis of MDA-MB-231 human breast cancer cells. Our results show significant induction of apoptosis in MDA-MB-231 cells incubated with JB. This effect was enhanced by combination with LY294002. In addition, treatment with JB could induce downregulation of the Bcl-2/Bax ratio, and subsequent promotion of mitochondrial release of cytochrome c and activation of caspase-3. Taken together, JB-induced apoptosis of MDA-MB-231 cells occurs through the mitochondrial pathway. Further, the PI3K/Akt signaling cascade plays a role in the induction of apoptosis in JB-treated cells. These observations suggest that JB may have therapeutic applications in the treatment of cancer.

The PI3K/Akt pathway is required for LPS activation of microglial cells

Upregulation of inflammatory responses in the brain is associated with a number of neurodegenerative diseases. Microglia are activated in neurodegenerative diseases, producing pro-inflammatory mediators. Critically, lipopolysaccharide (LPS)-induced microglial activation causes dopaminergic neurodegeneration in vitro and in vivo. The signaling mechanisms triggered by LPS to stimulate the release of pro-inflammatory mediators in microglial cells are still incompletely understood. To further explore the mechanisms of LPS-mediated inflammatory response of microglial cells, we studied the role of phosphatidylinositol 3-kinase (PI3K)/Akt signal transduction pathways known to be activated by toll-like receptor-4 signaling through LPS. In the current study, we report that the activation profile of LPS-induced pAkt activation preceded those of LPS-induced NF-κB activation, suggesting a role for PI3K/Akt in the pathway activation of NF-κB-dependent inflammatory responses of activated microglia. These results, providing the first evidence that PI3K dependent signaling is involved in the inflammatory responses of microglial cells following LPS stimulation, may be useful in preventing inflammatory based neurodegenerative processes.

Sericin can reduce hippocampal neuronal apoptosis by activating the Akt signal transduction pathway in a rat model of diabetes mellitus.

In the present study, a rat model of type 2 diabetes mellitus was established by continuous peritoneal injection of streptozotocin. Following intragastric perfusion of sericin for 35 days, blood glucose levels significantly reduced, neuronal apoptosis in the hippocampal CA1 region decreased, hippocampal phosphorylated Akt and nuclear factor kappa B expression were enhanced, but Bcl-xL/Bcl-2 associated death promoter expression decreased. Results demonstrated that sericin can reduce hippocampal neuronal apoptosis in a rat model of diabetes mellitus by regulating abnormal changes in the Akt signal transduction pathway.

Benefits of Recombinant Adeno-Associated Virus (rAAV)-Mediated Insulinlike Growth Factor I (IGF-I) Overexpression for the Long-Term Reconstruction of Human Osteoarthritic Cartilage by Modulation of the IGF-I Axis

Administration of therapeutic genes to human osteoarthritic (OA) cartilage is a potential approach to generate effective, durable treatments against this slow, progressive disorder. Here, we tested the ability of recombinant adeno-associated virus (rAAV)-mediated overexpression of human insulinlike growth factor (hIGF)-I to reproduce an original surface in human OA cartilage in light of the pleiotropic activities of the factor. We examined the proliferative, survival and anabolic effects of the rAAV-hIGF-I treatment in primary human normal and OA chondrocytes in vitro and in explant cultures in situ compared with control (reporter) vector delivery. Efficient, prolonged IGF-I secretion via rAAV stimulated the biological activities of OA chondrocytes in all the systems evaluated over extended periods of time, especially in situ, where it allowed for the long-term reconstruction of OA cartilage (at least for 90 d). Remarkably, production of high, stable amounts of IGF-I in OA cartilage using rAAV advantageously modulated the expression of central effectors of the IGF-I axis by downregulating IGF-I inhibitors (IGF binding protein [IGFBP]-3 and IGFBP4) while up-regulating key potentiators (IGFBP5, the IGF-I receptor and downstream mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 [MAPK/ERK-1/2] and phosphatidylinisitol-3/Akt [PI3K/Akt] signal transduction pathways), probably explaining the enhanced responsiveness of OA cartilage to IGF-I treatment. These findings show the benefits of directly providing an IGF-I sequence to articular cartilage via rAAV for the future treatment of human osteoarthritis.

Activation of protein kinase B (PKB/Akt) and risk of lung cancer among rural women in India who cook with biomass fuel

The impact of indoor air pollution (IAP) from biomass fuel burning on the risk of carcinogenesis in the airways has been investigated in 187 pre-menopausal women (median age 34years) from eastern India who cooked exclusively with biomass and 155 age-matched control women from same locality who cooked with cleaner fuel liquefied petroleum gas. Compared with control, Papanicolau-stained sputum samples showed 3-times higher prevalence of metaplasia and 7-times higher prevalence of dysplasia in airway epithelial cell (AEC) of biomass users. Immunocytochemistry showed up-regulation of phosphorylated Akt (p-Aktser473 and p-Aktthr308) proteins in AEC of biomass users, especially in metaplastic and dysplastic cells. Compared with LPG users, biomass-using women showed marked rise in reactive oxygen species (ROS) generation and depletion of antioxidant enzyme, superoxide dismutase (SOD) indicating oxidative stress. There were 2–5 times more particulate pollutants (PM10 and PM2.5), 72% more nitrogen dioxide and 4-times more particulate-laden benzo(a)pyrene, but no change in sulfur dioxide in indoor air of biomass-using households, and high performance liquid chromatography estimated 6-fold rise in the concentration of benzene metabolite trans,trans-muconic acid (t,t-MA) in urine of biomass users. Metaplasia and dysplasia, p-Akt expression and ROS generation were positively associated with PM and t,t-MA levels. It appears that cumulative exposure to biomass smoke increases the risk of lung carcinogenesis via oxidative stress-mediated activation of Akt signal transduction pathway.

Exogenous and autocrine growth factors stimulate human intervertebral disc cell proliferation via the ERK and Akt pathways

Intervertebral disc (IVD) degeneration is accompanied by growth factor-overexpression and increased cell proliferation, probably representing a tissue repair process. Accordingly, we studied the effect of exogenous and autocrine growth factors on the proliferation of human IVD cells. We observed that Platelet-Derived Growth Factor (PDGF), basic Fibroblast Growth Factor (bFGF), and Insulin-like Growth Factor-I (IGF-I) stimulate DNA synthesis of human IVD cells, through the activation of the MEK/ERK and the PI-3K/Akt signal transduction pathways. Furthermore, medium conditioned (CM) by IVD cells induced DNA synthesis in the same cells, indicating the secretion of autocrine growth factors. The MEK/ERK and PI-3K/Akt pathways were also induced by CM, while their inhibition reversed in large part the DNA synthesis induction by CM. These responses to the exogenous and autocrine growth factors were qualitatively similar in both nucleus pulposus (NP) and annulus fibrosus (AF) cell cultures. Immunohistochemical studies in human biopsies showed significant activation of both signaling pathways, which was most prominent in the clusters of proliferating cells. These in vitro and in vivo data indicate that the proliferation of human IVD cells is regulated by exogenous and autocrine growth factors mainly via the MEK/ERK and PI-3K/Akt pathways; this may contribute to the design of future interventional approaches.

Blockage of CGEN-928 Expression Down-Regulates AKT Signaling Pathway and Inhibits Multiple Myeloma (MM) Cell Proliferation

Abstract 5102We have recently reported that CGEN-928 is highly expressed on the cell membrane of cell lines, human xenografts, and primary tumor cells from MM. Anti-CGEN-928 (anti-TM21) polyclonal antibody blocked the expression of CGEN-928 which decreased MM tumor cell proliferation and increased apoptosis in the MM cell lines MM1s, RPMI8226 and U266 as well as primary MM tumor cells. The mechanism through which blocking CGEN-928 decreases MM tumor cell proliferation and enhances apoptosis has not been elucidated clear. In this study, a CGEN-928 shRNA (lentiviral particles) was used to silence this gene’s expression, and determine its impact on the AKT signal transduction pathway which has been shown to play an important role in MM tumor cell metabolism proliferation, and survival. Briefly, MM1s or primary MM tumor cells were cultured in a 12-well plate for 24 hours prior to the viral infection. On the following day, a mixture of 5ug/ml Polybrene and fresh medium were added to the cells. The CGEN-928 shRNA lentiviral particles were then added to the culture. While transducing cells, we treated a portion of the cells with a negative control through introduction of control shRNA lentiviral particles. To ensure we achieved a successful transduction, we also treated another portion of the cells with cop GFP control Lentiviral particles. We confirmed that 75% of MM cells were transduced based on GFP+ cell counts after 24 hours treatment. The day following the transduction, the cultured medium was removed and replaced with fresh medium without polybrene. Two days following transduction, we used fresh 10ug/ml puromycin-containing medium to select stable MM cells. We replaced the medium with fresh puromycin-containing medium every three days until resistant MM tumor cells were stable. Proliferation rate of the MM1s tumor cells transduced with CGEN-928 shRNA (85%) 24 hours was much lower than the tumor cells transduced with control lentiviral particles rate (170%). The proportion of MM cells undergoing apoptosis treated with CGEN-928 shRNA (42%) was higher than MM cells transduced with control lentiviral particles (13%). We next examined several protein phosphorylation sites related to AKT signaling pathway by Western blot. The results showed AKT1 phosphorylation in MM tumor cells transduced with CGEN-928 shRNA or anti- CGEN-928 polyclonal antibody was decreased and phosphorylation of c-Raf, GSK-3β, factors downstream of AKT were also down-regulated. PTEN phosphorylation slightly decreased in MM cell treated with anti-CGEN-928 antibody but did not change in MM cells silenced with CGEN-928 shRNA. We further examined downstream gene expression of the AKT pathway when CGEN-928 was silenced using siRNA or the anti-CGEN-928 TM-21 antibody. We found AKT1 gene expression was reduced in the presence of CGEN-928 siRNA or antibody but it did not impact ATK2 and AKT3. mTOR gene expression in MM tumor cells was decreased with exposure to CGEN-928 siRNA but anti-TM21 showed no effect. Cyclin D1 gene expression in MM tumor cell was not affected by CGEN-928 siRNA and antibody. These studies suggest that blockage of CGEN-928 antigen expression inhibits MM tumor cell proliferation and enhance tumor cell apoptosis through AKT signaling pathway. Currently, a monoclonal anti-CGEN-928 antibody is in development that will be used by our group to evaluate its anti-MM effects both in vitro and in vivo using our SCID-hu models of human MM. Disclosures:Berenson:Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding, Speakers Bureau; Onyx Pharmaceuticals: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Medtronic: Consultancy, Honoraria, Research Funding, Speakers Bureau; Merck: Research Funding; Genentech: Research Funding.

The Effect of Eltrombopag on Platelet Resistance to Apoptosis: The Role of the Bcl-Xl Pathway

Abstract 1158 INTRODUCTION:Immune thrombocytopenia (ITP) is typically characterized by increased platelet destruction and reduced platelet production. Eltrombopag and Romiplostim are thrombopoietin receptor (TPO-R) agonists that are known to increase platelet counts in patients with ITP by stimulating thrombopoiesis. Platelets also express TPO-R on their surface, but it is unknown whether the thrombopoietin mimetics (TPO-M) have a direct effect on the circulating platelets. Although controversial, in a very small number of ITP patients, TPO-M agents may increase platelet counts in 2–5 days, earlier than would be expected from de novo megakaryocytopoiesis. Platelet survival is hypothesized to be mediated by two molecular intermediates in an apoptotic pathway, Bcl-xL and Bak. Bcl-xL/Bak protein expression in megakaryocytes is regulated in part by TPO-mediated activation of Akt pathways through Jak2 and Stat5. We hypothesized that an increase in platelet count in the first week of treatment might be mediated by TPO-R signaling, resulting in decreased platelet apoptosis. This study explored whether Eltrombopag or Romiplostim treatment has anti-apoptotic effects on platelets of patients with ITP. METHODS:Following a treatment wash out period, 75 mg of Eltrombopag once daily or 10 mcg/kg weekly of Romiplostim was initiated for 2 weeks. Blood counts were measured on days 1, 3, 5, 8, 10, 12, and 15. Platelet function and survival was assessed on days 1, 8, and 15 by: immature platelet fraction (IPF), glycocalicin index, Bcl-xL inhibitor (ABT-737) assay, measurement of Bcl-xL by western blot, measurement of several members of the Bcl-xL Akt mediated, apoptotic pathway by flow cytometry (FACS), bleeding score, measurement of thrombin-anti-thrombin complexes (TATs), and quantification of microparticles. RESULTS:Eight of 10 patients responded to treatment with Eltrombopag with a platelet count ≥ 50,000/μL, and 6 of the 8 responders at least doubled their counts during the 2 weeks of treatment. All 3 patients treated with Romiplostim responded with platelet count ≥ 50,000/μL. In both treatment groups there was a significant increase in median platelet count (p<0.001), median large platelet count (p<0.01), and median absolute IPF (A-IPF, p<0.01), while there was no significant change in median % IPF. The dose of ABT-737 required to kill half of the platelets in the sample (IC50) in the Eltrombopag group was lower in patients at day 1 than in non-ITP controls, and there was an increase in resistance to apoptosis between days 1 and 8, but these changes did not reach statistical significance. Between days 8 and 15 the IC50 declined to pre-treatment levels. In the Romiplostim group there was no significant difference in IC50 between the control and the patients over the 2 weeks of study. There was no significant correlation between the platelet counts and the IC50 values. FACS analysis of members of the AKT signal transduction pathway revealed increased activation of each of the markers between days 1 and 8, followed by a decrease between days 8 and 15. The levels of Bcl-xL and phosphor-AKT(308) decreased from day 1 to day 15. The other lab tests are pending. DISCUSSION:Because the A-IPF increased by less than the platelet increase and because the lifespan of the A-IPF is not known, it is unclear if the platelet count increase is solely a result of increased platelet production. Platelet lifespan may be enhanced by Eltrombopag treatment as there was a parallel albeit transient increase in AKT activation markers and platelet apoptosis resistance in the Eltrombopag group. Treatment with Romiplostim did not appear to affect apoptosis resistance although it did result in transient AKT activation. Our data suggest that platelets are more resistant to apoptosis when the levels of anti-apoptotic factors (eg. PTEN, Phospho-GSK3β) involved in the AKT/Bcl-xL pathway are greatest despite a concomitant increase in pro-apoptotic factors (eg. Bak, Bax). Since both the increased AKT activation and apoptotic resistance returned to baseline at day 15, megakaryocytes and platelets already present at the start of treatment may respond differently than those generated de novo in the presence of TPO mimetics. [Display omitted] Disclosures:Bussel:Portola: Consultancy; Eisai: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; GlaxoSmithKline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Cangene: Research Funding; Genzyme: Research Funding; Immunomedics: Research Funding; Ligand: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Shionogi: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sysmex: Research Funding.

CCN3 increases cell motility and MMP-13 expression in human chondrosarcoma through integrin-dependent pathway

Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. CCN3, also called nephroblastoma overexpressed gene (NOV), regulates proliferation and differentiation of cancer cells. However, the effect of CCN3 on migration activity in human chondrosarcoma cells is mostly unknown. Here, we found that CCN3 increased the migration and expression of matrix metalloproteinase (MMP)-13 in human chondrosarcoma cells (JJ012 cells). αvβ3 or αvβ5 monoclonal antibody (mAb), phosphatidylinositol 3-kinase (PI3K) inhibitors (Ly294002 and wortmannin) and Akt inhibitor inhibited the CCN3-induced increase of the migration and MMP-13 upregulation of chondrosarcoma cells. CCN3 stimulation increased the phosphorylation of focal adhesion kinase (FAK), PI3K, and Akt. In addition, NF-κB inhibitors also suppressed the cell migration and MMP-13 expression enhanced by CCN3. Moreover, CCN3 increased NF-κB luciferase activity and binding of p65 to the NF-κB element on the MMP-13 promoter. Taken together, our results indicate that CCN3 enhances the migration of chondrosarcoma cells by increasing MMP-13 expression through the αvβ3/αvβ5 integrin receptor, FAK, PI3K, Akt, p65, and NF-κB signal transduction pathway.

Arteriogenic Therapy by Intramyocardial Sustained Delivery of a Novel Growth Factor Combination Prevents Chronic Heart Failure

Therapeutic angiogenesis is a promising approach for the treatment of cardiovascular diseases, including myocardial infarction and chronic heart failure. We aimed to improve proangiogenic therapies by identifying novel arteriogenic growth factor combinations, developing injectable delivery systems for spatiotemporally controlled growth factor release, and evaluating functional consequences of targeted intramyocardial growth factor delivery in chronic heart failure. First, we observed that fibroblast growth factor and hepatocyte growth factor synergistically stimulate vascular cell migration and proliferation in vitro. Using 2 in vivo angiogenesis assays (n=5 mice per group), we found that the growth factor combination results in a more potent and durable angiogenic response than either growth factor used alone. Furthermore, we determined that the molecular mechanisms involve potentiation of Akt and mitogen-activated protein kinase signal transduction pathways, as well as upregulation of angiogenic growth factor receptors. Next, we developed crosslinked albumin-alginate microcapsules that sequentially release fibroblast growth factor-2 and hepatocyte growth factor. Finally, in a rat model of chronic heart failure induced by coronary ligation (n=14 to 15 rats per group), we found that intramyocardial slow release of fibroblast growth factor-2 with hepatocyte growth factor potently stimulates angiogenesis and arteriogenesis and prevents cardiac hypertrophy and fibrosis, as determined by immunohistochemistry, leading to improved cardiac perfusion after 3 months, as shown by magnetic resonance imaging. These multiple beneficial effects resulted in reduced adverse cardiac remodeling and improved left ventricular function, as revealed by echocardiography. Our data showing the selective advantage of using fibroblast growth factor-2 together with hepatocyte growth factor suggest that this growth factor combination may constitute an efficient novel treatment for chronic heart failure.

Effect of PKB/Akt signal pathway in mifepristone-induced apoptosis in prostate cancer cell line PC-3

Objective To investigate the cell apoptosis induced by mifepristone in prostate carcinoma cell line PC-3 and the mechanisms involved. Methods Western blotting analysis was used to detect Akt phosphorylation and associated protein expression in different prostate cancer cell line cells and PC-3 cells treated with mifepristone. Results In PC-3 cells, Akt phosphorylation, that was p-Akt (Thr308) and p-Akt ( Ser473 ) , was significantly higher than the hormone-dependent prostate cancer LNCaP cells, and their expression levels were respectively 0. 27 ±0. 05 and 0. 22 ±0.07, 0.46 ±0.09 and 0. 37 ±0. 10 (P ＜0.05). The p-Akt (Thr308) and p-Akt (Ser473) phosphorylation in PC-3 cells treated with 10 μmol/L mifepristone was significantly decreased. The p-Akt ( Thr308) phosphorylation was particularly marked, its expression was respectively 0. 24 ± 0. 08, 0. 11 ± 0. 03, 0. 05 ± 0. 01 and 0. 01 ± 0. 00 during 4-24 h, and declined rapidly as compared with the p-Akt ( Ser473) phosphorylation which expression levels were respectively 0. 56 ± 0. 17, 0.42 ± 0. 11, 0. 28 ± 0. 09 and 0. 12 ± 0. 05 during 4-24 h. Caspase-9 and caspase-3 were activated into cleavage fragment in the PC-3 cells treated with mifepristone. Conclusion Akt kinase plays an important role in prostate cancer growth, development and hormone-dependent to hormone-independent transformation. The p-Akt ( Thr ) phosphorylation, which plays a dominant role in Akt phosphorylation , is necessary in the course of Akt activation. Mifepristone inhibits prostate cancer PC-3 cell proliferation and induces apoptosis by suppressing the Akt signal transduction pathway and activating caspase. Key words: Prostate carcinoma; Mifepristone; Apoptosis; Akt/PKB signal pathway

Abstract 1201: Role of the chemokine CXCL12 in the estrogen response of breast cancer cells

Abstract The purpose of this study was to investigate the extent to which CXCL12 mediates the effects of estrogen in estrogen-responsive breast cancer cells. Expression microarray studies have identified CXCL12 as a gene regulated consistently by estrogen in estrogen-responsive breast cancer cells in our own and other laboratories and it has been reported that CXCL12 mediates the proliferative effects of estrogen. CXCL12 activates two seven transmembrane G protein-coupled receptors, CXCR4 and CXCR7, that transducer signals via heterotrimeric G-proteins. CXCR4 has long been accepted, whereas CXCR7 was identified more recently as a potential CXCL12 receptor. Methods: Expression of CXCL12, CXCR4 and CXCR7 was measured in five estrogen-responsive and five estrogen-nonresponsive breast cancer cell lines by qRT-PCR and western transfer analysis. The effects of CXCL12 on proliferation of the cells were assessed by DNA quantification and on migration in a modified Boyden chamber assay. The ability of CXCL12 to activate the downstream signalling molecules, MAPK and Akt, was measured by western transfer analysis with a phospho-specific antibodies. The motogenic responses of different breast cancer cell lines to CXCL12 were measured in modified Boyden chamber and in vitro wounding assays. The relative importance of CXCR4 in the CXCL12 response was probed with a specific inhibitor of CXCR4 receptor. Results: The expression of CXCL12, CXCR4 and CXCR7 varied greatly between the ten breast cancer cell lines analyzed. CXCL12 expression was highest in estrogen-responsive T-47D cells whereas CXCR4 and CXCR7 expression was highest in estrogen-nonresponsive MDA-MB-231 cells. CXCL12 was detected after secretion into medium conditioned by T-47D and MCF-7 breast cancer cells. CXCL12 did not stimulate the proliferation of MCF-7 or T-47D cells in the absence or presence of estrogen. CXCL12 did stimulate breast cancer cell migration in a concentration-dependent manner and cells that express high concentrations of the CXCL12 receptor were more responsive to CXCL12. CXCL12 stimulated Phosphorylation of both MAPK and Akt in estrogen-responsive and non-responsive breast cancer cells. Conclusion: This study shows that CXCL12 does not mediate the proliferative effects of estrogen on breast cancer cells. CXCL12 does however stimulate the migration of both estrogen-responsive and estrogen non-responsive breast cancer cells. CXCL12 mediates it effects in breast cancer cells by activation on both the MAPK and Akt signal transduction pathways. 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 1201. doi:10.1158/1538-7445.AM2011-1201

Abstract 1392: CTGF inhibits cell motility in oral cancer cells through reducing COX-2 expression

Abstract Oral squamous cell carcinoma (SCC) has a striking tendency to migrate and metastasize. Cyclooxygenase (COX)-2, the inducible isoform of prostaglandin synthase, has been implicated in tumor metastasis. Connective tissue growth factor (CTGF), a secreted protein that binds to integrins, modulates the invasive behavior of certain human cancer cells. However, the effect of CTGF on migration activity and COX-2 expression in human oral cells is mostly unknown. Here we found that CTGF reduced the migration and expression of COX-2 in human oral cancer cells. αvβ5 monoclonal antibody (mAb), phosphatidylinositol 3-kinase inhibitor (PI3K; Ly294002 and wortmannin) and Akt inhibitor reversed the CTGF-inhibited the migration and COX-2 down-regulation of oral cancer cells. CTGF stimulation decreased the phosphorylation of focal adhesion kinase (FAK), PI3K and Akt. In addition, c-Jun siRNA also antagonized the CTGF-inhibited migration and COX-2 expression. Moreover, CTGF decreased the binding of c-Jun to the AP-1 element on the COX-2 promoter. Taken together, our results indicated that CTGF inhibits the migration of oral cancer cells by decreasing COX-2 expression through the αvβ5 integrin receptor, FAK, PI3K, Akt, c-Jun and AP-1 signal transduction pathway. 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 1392. doi:10.1158/1538-7445.AM2011-1392

Abstract 1399: CCN3 enhances migration and ICAM-1 upregulation via avb3 integrin, ILK, Akt, and NF-kB-dependent pathway in human prostate cancer cells

Abstract Nephroblastoma overexpressed (NOV/CCN3), from the CCN gene family, is a secreted and matrix-associated protein, which is involved in many cellular functions such as growth, differentiation and adhesion. However, the effect of CCN3 in human prostate cancer is still unclear. Here, we found that CCN3 and intercellular adhesion molecule-1 (ICAM-1) expression level were related to cell migration ability in prostate cancer cells. In addition, CCN3 increased the migration and expression of ICAM-1 in human prostate cancer cells. RGD peptide, αvβ3 monoclonal antibody, integrin-linked kinase (ILK) and Akt inhibitors but not RAD peptide inhibited the CCN3-induced increase of the migration and ICAM-1 upregulation of prostate cancer cells. CCN3 stimulation activated the ILK/Akt/ NF-κB pathway. Moreover, CCN3 increased the binding of p65 to the NF-κB element on the ICAM-1 promoter. Finally, knock down of CCN3 expression markedly inhibited cell migration ability, tumor growth and bone destruction. Taken together, our results indicated that CCN3 enhances the migration of prostate cancer cells by increasing ICAM-1 expression through the αvβ3 integrin receptor/ ILK/ Akt and NF-κB signal transduction pathway. Therefore, CCN3 may be developed as alternate therapy of prostate cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1399. doi:10.1158/1538-7445.AM2011-1399