Pro Survival Research Articles

Dual Regulation of Cell Death and Cell Survival upon Induction of Cellular Stress by Isopimara-7,15-Dien-19-Oic Acid in Cervical Cancer, HeLa Cells In vitro.

The Fritillaria imperialis is an ornamental flower that can be found in various parts of the world including Iraq, Afghanistan, Pakistan, and the Himalayas. The use of this plant as traditional remedy is widely known. This study aims to unveil the anti-cancer potentials of Isopimara-7,15-Dien-19-Oic Acid, extracted from the bulbs of F. imperialis in cervical cancer cell line, HeLa cells. Flow cytometry analysis of cell death, gene expression analysis via cDNA microarray and protein array were performed. Based on the results, Isopimara-7,15-Dien-19-Oic acid simultaneously induced cell death and promoted cell survival. The execution of apoptosis was apparent based on the flow cytometry results and regulation of both pro and anti-apoptotic genes. Additionally, the regulation of anti-oxidant genes were up-regulated especially thioredoxin, glutathione and superoxide dismutase- related genes. Moreover, the treatment also induced the activation of pro-survival heat shock proteins. Collectively, Isopimara-7,15-Dien-19-Oic Acid managed to induce cellular stress in HeLa cells and activate several anti- and pro survival pathways.

Targeting Cancer Stem Cell Survival in Plasma Cell Leukemia with a Pan-BCL2 Inhibitor

INTRODUCTIONRepresenting ten percent of all hematologic malignancies, multiple myeloma (MM) is typified by clonal plasma cell proliferation in the bone marrow (BM) and may progress to a therapy-resistant stage characterized by circulating malignant plasma cells, which is termed plasma cell leukemia, (PCL). Notably, copy number amplification involving the myeloid cell leukemia (MCL)-1 locus, and translocations at the BCL2 locus have been described in high-risk MM. Moreover, BCL2 family members, including both MCL1 and BCL2, are highly expressed in relapsed MM. Finally, downregulation of BCL2 family members was reported to increase sensitivity to lenalidomide, a standard treatment for MM, suggesting that BCL2-targeted treatments may obviate therapeutic resistance.Sabutoclax, a potent small molecule inhibitor of 6 anti-apoptotic BCL2 family proteins, was shown to inhibit cancer stem cell (CSC) survival in chronic myeloid leukemia (CML) (Goff et al Cell Stem Cell 2013). Thus, we investigated BCL2 pro-survival splice isoform expression in therapy-resistant MM and PCL and evaluated whether Sabutoclax reduces malignant plasma cell burden in humanized primagraft assays.METHODS AND RESULTSBCL2 pro survival expression in primary myeloma samplesMononuclear cells from MM or PCL samples were isolated by Ficoll-Paque density gradient separation and collected for RNA extraction or FACS Aria purified into CD138+ or CD34-/CD138- cancer stem cells (CSC) subsets. To evaluate BCL2 pro-survival family member expression, splice isoform-specific quantitative PCR was performed to measure expression of pro-survival long isoforms compared with short splice isoforms, which are pro-apoptotic. Interestingly, MM and PCL patients displayed higher levels of BCL2-L, MCL1-L, BCLX-L and BLF1-L compared to normal controls. Moreover, prolonged lenalidomide exposure increased BCL2-L and MCL1-L expression in the myeloma cell line H929, compared to untreated cells.Sabutoclax Treatment of a Novel Humanized Plasma Cell Leukemia Primagraft ModelMononuclear cells from three primary PCL patient samples were stably transduced with a GFP-luciferase lentiviral vector and transplanted intrahepatically in newborn RAG2γ-/- c-/- mice. Engraftment was monitored by peripheral blood free light chain ELISA assays. Flow cytometric analyses revealed robust engraftment of PCL cells in bone marrow, spleen, liver and peripheral blood. Once transplanted mice displayed significant tumor burden above background free light chain levels, animals were randomized by ELISA values in vehicle versus Sabutoclax groups. Sabutoclax was selected because this pan-BCL2 targeted compound, unlike related BCL2 inhibitors such as ABT-199, also inhibits MCL1. Sabutoclax (10mg/kg) was administered intravenously twice weekly for two to four doses. Sabutoclax treated PCL mice showed reduced human plasma cell burden in bone marrow and spleen tissues compared to vehicle controls.CONCLUSIONExpression of pro-survival BCL2 splice isoforms, including BCL2-L and MCL1-L, portends PCL engraftment in immunocompromised mice. Treatment of human PCL engrafted mice with Sabutoclax reduces malignant plasma cell survival in hematopoietic tissues. Thus, selective targeting of pro-survival isoform expressing CSC with a pan-BCL2 inhibitor may abolish BCL2 and MCL1-dependent therapeutic resistance in MM and PCL. DisclosuresJamieson:GlaxoSmithKline: Research Funding; Johnson & Johnson: Research Funding.

Novel Mechanisms of Growth Inhibition By Histone Deacetylase Inhibitors in MPN

Histone deacetylase inhibitors (HDACi) are being evaluated in several clinical trials for the treatment of hematological malignancies and 2 of them have been approved for the treatment of T cell lymphoma. We have investigated the effectiveness of using 2 HDACi - Vorinostat (SAHA) and Panobionstat for treatment of systemic mastocytosis (SM) for which no treatments are available. SAHA and Panobinostat showed concentration dependent inhibition of proliferation in mastocytosis patient derived cell lines, HMC1.1 and HMC1.2 bearing either single mutation in the juxtamembrane domain KITG560V or double mutation also including the activation loop KITG560V,D816V, respectively. Interestingly HMC1.2 cells with 2 mutations in KITG560V, D816V were more sensitive to treatment with both SAHA (IC50 ~ 260 nM) and Panobinostat (IC50 ~11 nM) as compared to HMC1.1 cells (IC50 ~ 600 nM for SAHA and IC50 ~ 23 nM with Panobionstat) that have a single mutation in KIT. The inhibition of proliferation by HDACi treatment appeared to be associated with modulation of cell cycle and differentiation rather than induction of apoptosis. We further tested the effect of combining HDACi treatment with tyrosine kinase inhibitor, Dasatinib. Combination of Dasatinib (5 - 20 nM) with either SAHA or Panbionostat was more effective than any of the single drugs in HMC1.1 cells. However such a combination effect was not seen in HMC1.2 cells. Over 90% patients with aggressive systemic mastocytosis carry KITD816V mutation. This particular mutation of KIT is highly resistant to therapeutics and is associated with poor survival. Thus patients carrying the KITD816V mutation are likely to benefit from treatment with HDACi while juxtamembrane mutation bearing patients would benefit more from a combination of HDACi with kinase inhibitors such as Dasatinib. To further understand the mechanism behind HDACi inhibition of KITD816V mediated oncogenic signals, we expressed KITD814V (murine equivalent of human D816V) in myeloid cells and treated them with SAHA and Panobinostat. Cells expressing KITD814V showed cytokine independent proliferation and interestingly, cytokine independent proliferation was more sensitive to inhibition by both SAHA and Panbinostat as compared to the cytokine dependent proliferation. In contrast to HMC cells, inhibition of cytokine independent proliferation by HDACi in KITD814V oncogene expressing cells was associated with significant increase in apoptosis. The percent cells negative for Annexin V stain decreased from 74% in control to ~46% in 24h upon treatment with 500 nM SAHA or 10 nM Panbinostat (p< 0.001; n=3). Induction of apoptosis by HDACi in KITD814V oncogene expressing cells was significantly suppressed in presence of cytokine. Consistent with the in vitro data, treatment with SAHA prolonged the survival of mice transplanted with myeloid cells expressing KITD814V mutation as compared to the vehicle treated group (Median survival - 21d (veh), 34d (SAHA), p=0.0015, n=5). In addition to increased histone acetylation, treatment of cells with HDACi also leads to increase in acetylation of chaperone proteins such as HSP90 which can mediate proteasomal degradation of oncogenes and has been suggested as one of the mechanisms by which HDACi mediates growth inhibition. In contrast, we found no significant decrease in mutant KIT protein expression or AKT expression and phosphorylation upon treatment with either of the HDACi. Though expression of Pim1 a pro survival serine/threonine kinase was down-regulated by SAHA and Panobinostat. Treatment with HDACi was also associated with differential modulation of STAT3, STAT5 and ERK pathways. Treatment with both SAHA and Panobinostat down-regulated STAT5 phosphorylation and up-regulated ERK1/2 phosphorylation with more increase in ERK1/2 phosphorylation observed in cells with higher sensitivity to inhibition by HDACi. Though increased activation of the ERK pathway has been associated with leukemogenesis; there is some evidence to indicate that hyper activation of ERK pathway in absence of additional leukemogenic signals activates a pro senescence program. Differential expression of Pim1 and along with differential modulation of STAT and ERK pathways may contribute to differential sensitivity of the cells to HDACi and are likely to be of therapeutic value in aggressive systemic mastocytosis that has so far been associated with poor prognosis. DisclosuresNo relevant conflicts of interest to declare.

Quercetin in brain diseases: Potential and limits

Quercetin is a ubiquitous flavonoid present in beverages, food and plants that has been demonstrated to have a role in the prevention of neurodegenerative and cerebrovascular diseases.In neuronal culture, quercetin increases survival against oxidative insults. Antioxidation appears to be a necessary but not sufficient condition for its neuroprotective action and modulation of intracellular signaling and transcription factors, increasing the expression of antioxidant and pro survival proteins and modulating inflammation, appears as important for neuronal protection. Quercetin also regulates the activity of kinases, changing the phosphorylation state of target molecules, resulting in modulation of cellular function and gene expression. Concentrations of quercetin higher than 100 μM consistently show cytotoxic and apoptotic effects by its autoxidation and generation of toxic quinones.In vivo, results are controversial with some studies showing neuroprotection by quercetin and others not, requiring a drug delivery system or chronic treatments to show neuroprotective effects. The blood and brain bioavailability of free quercetin after ingestion is a complex and controversial process that produces final low concentrations, a fact that has led to suggestions that metabolites would be active by themselves and/or as pro-drugs that would release the active aglycone in the brain. Available studies show that in normal or low oxidative conditions, chronic treatments with quercetin contributes to re-establish the redox regulation of proteins, transcription factors and survival signaling cascades that promote survival. In the presence of highly oxidative conditions such as in an ischemic tissue, quercetin could become pro-oxidant and toxic. At present, evidence points to quercetin as a preventive molecule for neuropathology when administered in natural matrices such as vegetables and food. More research is needed to support its use as a lead compound in its free form in acute treatments, requiring new pharmaceutical formulations and/or structural changes to limit its pro-oxidant and toxic effects.

Effect of C6Ceramide on chemotoxicity of gemcitabine against chemoresistant pancreatic cell lines.

e15286 Background: Pancreatic cancer is a highly chemo-resistant malignancy with 5% overall survival, and limited treatment options with chemotherapy (Gemcitabine) and surgical resection possible in < 20% of patients with post resection survival of only 15-20%. Our studies suggest potential value of C6 Ceramide as a chemo enhancing biologic in therapy of chemoresistant pancreatic cancer. C6 Ceramide is an active sphingomyelin end product that has the major biologic function of inducing apoptosis in cells compromised or damaged by injury or malignancy, and appears to have potential value in therapy of malignancy. We have demonstrated C6 Ceramide inhibition of pro survival (AKT/PI3K/mTOR) and KRAS mutant pathways, which promote pancreatic cancer growth and metastases. Methods: Cell lines obtained from 4 freshly cultured pancreatic cancers, and 3 well established cancer cell lines (ATCC) were cultured in 96 well plates, 4000 cells/well, and were treated at 24 hours with Gemcitabine in 0.9% NaCl (1,2,5,10 ug/...

Peptide-derived from Seahorse Exerts a Protective Effect against Cholinergic Neuronal Death in in vitro Model of Alzheimer's Disease

Hippocampus trimaculatus are highly valued and the most heavily traded seahorse species for traditional medicine purposes in many Asian countries. One of the main interesting features in H.trimaculatus is their richness in protein which can be hydrolyzed into bioactive peptides. In the previous study, H. trimaculatus was hydrolyzed using commercial enzymes, the peptides responsible for neuroprotective activity was identified as HTP-1. In the present study, in vitro co-culture system of neuronal (PC12) cells and Aβ42 oligomer-stimulated murine microglia cells (BV2 cells) were used to test neuroprotective effects of HTP-1. The co-culture system showed that HTP-1 protected PC12 cells from BV2 neurotoxic responses. In addition, the activation of PI3K/Akt signaling pathway by HTP-1 were confirmed. The PI3K/Akt activation was found to be mediated through transforming growth factor-β (TGF-β) induction by HTP-1. Furthermore, this signaling pathway was found to up-regulate the expression of pro survival protein expression in PC12 cells. Collectively, HTP-1 has potent protective effect against neuronal cells death in in vitro model of Alzheimer's disease.

ASTROCYTES IN THE NEUROPROTECTION AFTER BRAIN STROKE

Event Abstract Back to Event ASTROCYTES IN THE NEUROPROTECTION AFTER BRAIN STROKE Andrea Del Pilar Becerra Calixto1 and Gloria Patricia Cardona Gomez1* 1 University of Antioquia, Group of Neuroscience of Antioquia, Colombia Astrocytes are specialized glial cells of the nervous system, which have multiple homeostatic functions for the survival and maintenance of the neurovascular unit. It has been shown that astrocytes have critical role in the dynamics pro survival conferring neuroprotective, angiogenic, immunomodulatory, neurogenic, antioxidants and regulatory synapse functions (Shen et al 2012; Gimsa et al 2013; Proschel et al 2014); making them excellent candidates as the source of neuroprotection and neurorestauration of tissue affected by events ischemia and / or reperfusion. However, these cells also may be involved in negative responses such as reactive astrocytes and glial scar under chronic excitotoxic responses generated by these events. To know what are the key points in the pro and anti-survival responses of astrocytes, would allow use them as targets in cellular therapies. This review has aim to study the mechanisms for neuroprotection in these cells (Posada-Duque et al submitted), which would make them targets of cell therapy, through of inducing regeneration, such as vehicle for corrective molecular systems and trigger endogenous cellular events that can recover the tissue homeostasis, which is lost after progressive damage. Figure 1 Acknowledgements The research reported in this publication was supported by CODI # 214-970 and Colciencias project #111554531400. References Gims, U., Mitchison, N. A., Brunner-Weinzierl, M. C. (2013) Immune privilege as an intrinsic CNS property: astrocytes protect the CNS against T-cell-mediated neuroinflammation. Mediators Inflamm 2013, 320519. doi: 10.1155/2013/320519. Posada-Duque, R., Palacio-Castañeda, V., Cardona-Gómez, GP. CDK5 knockdown Astrocytes are trophic source for neuronal protection through Rac1. Submitted. Proschel, C., Stripay, J. L., Shih, C. H., Munger, J. C., Noble, M. D. (2014) Delayed transplantation of precursor cell-derived astrocytes provides multiple benefits in a rat model of Parkinsons. EMBO Mol Med 6, 504-518. doi:10.1002/emmm.201302878. Shen Y. , Sun, A., Wang, Y., Cha, D., Wang, H., Wang, F., Feng, L., Fang, S. (2012) Upregulation of mesencephalic astrocyte-derived neurotrophic factor in glial cells is associated with ischemia-induced glial activation. J Neuroinflammation 9, 254 doi:10.1186/1742-2094-9-254. Keywords: Astrocytes, cell therapy, Neuroprotection, cerebral ischemia, excitotoxicity Conference: Latin-American School on glial cells in the diseased brain (IBRO), Bogotá, Colombia, 13 Jul - 17 Jul, 2015. Presentation Type: Oral Presentation Topic: Traumatic and Cerebrovascular diseases Citation: Becerra Calixto A and Cardona Gomez G (2015). ASTROCYTES IN THE NEUROPROTECTION AFTER BRAIN STROKE. Conference Abstract: Latin-American School on glial cells in the diseased brain (IBRO). doi: 10.3389/conf.fncel.2015.35.00007 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 10 Mar 2015; Published Online: 16 May 2015. * Correspondence: Prof. Gloria Patricia Cardona Gomez, University of Antioquia, Group of Neuroscience of Antioquia, Medellin, Antioquia, 05001000, Colombia, patricia.cardona@neurociencias.udea.edu.co Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Andrea Del Pilar Becerra Calixto Gloria Patricia Cardona Gomez Google Andrea Del Pilar Becerra Calixto Gloria Patricia Cardona Gomez Google Scholar Andrea Del Pilar Becerra Calixto Gloria Patricia Cardona Gomez PubMed Andrea Del Pilar Becerra Calixto Gloria Patricia Cardona Gomez Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Overcoming Resistance to Apoptosis in Multiple Myeloma By Simultaneous Inhibition of Bcl2 and IAP Families of Anti-Apoptotic Proteins

Background: Multiple myeloma (MM) cells evade apoptosis through multiple mechanisms thus enabling it to evade therapy. The Bcl2 family of anti-apoptotic proteins is aberrantly expressed in MM cell lines and patient cells. Yet, pharmacological intervention of this family appears to have significant activity only in molecular subgroups of MM patients. This clearly suggests alternate mechanisms of overcoming apoptotic signals in MM cells in addition to the Bcl2 family, through proteins such as IAPs. We have previously shown that simultaneous inhibition of the three major IAP proteins, namely cIAP1, cIAP2 and XIAP is required to induce pronounced apoptosis in MM cells. However, IAP inhibition results in apoptosis in only some MM cell lines and patient cells. Given that levels of Bcl2 family proteins are unaffected by IAP inhibition, we hypothesized that combined inhibition of the IAP proteins using a SMAC mimetic LCL161 and the Bcl2 family proteins using a pan-Bcl2 inhibitor obatoclax (OBX) will lead to more pronounced and synergistic cell death in a broader subgroup of MM patients.Methods: LCL161 was synthesized by Novartis Inc. (Basel, Switzerland). OBX was purchased from Selleckchem (Houston, USA). Stock solutions were made in DMSO, and subsequently diluted in RPMI-1640 medium for use. MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum (20% serum for primary patient cells) supplemented with L-Glutamine, penicillin, and streptomycin. Cytotoxicity was measured using the MTT viability assay and proliferation using thymidine uptake. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI) for cell lines and patient cells. Immunoblotting was done on cell extracts at various time points following incubation with the drugs in order to study the cell signaling pathways and aResults: LCL161/OBX combination induced synergistic cytotoxicity and anti-proliferative effects on a broad range of human MM cell lines, including drug resistant cell lines like DOX40 and MM1R. Components of the bone marrow microenvironment including bone marrow stromal cells and tumor promoting cytokines (VEGF, IGF and IL6) were unable to protect MM cells from the effects of the drug combination. We saw a time dependent increase in apoptosis, with the combination inducing significantly more apoptosis than either of the single agents alone. Examining the mechanism of action of the drug combination showed clear inhibition of the IAP proteins, activation of caspases 9, 8, 3 and Bid by LCL161 and the combination and up regulation of the pro-apoptotic proteins Bim, Bid, Puma and Noxa and accumulation of LC3-II by OBX and the combination. Using chloroquine along with the OBX, we were able to demonstrate that OBX induced protective autophagy and the addition of LCL161 was able to overcome this protective effect induced after single agent OBX treatment. Since protective autophagy can be induced by the ER stress response, we then examined the expression levels of proteins involved in this pathway. We observed clear induction of ER stress mediated UPR pathway by both the drugs. However, LCL161 and OBX induced different branches of the UPR pathway. OBX activated the ATF6 and pErk/peif2α/ATF4 branches of the UPR, both of which have been implicated in cell survival during ER stress. ATF4 under irrecoverable ER stress can lead to increase in transcription of CHOP and cause apoptosis. We therefore examined levels of CHOP and observed no induction of CHOP post treatment with either of the drugs or the combination. LCL161, however differentially modulated the IRE1 branch of the UPR by down regulating Xbp-1 splicing, which is a pro survival activity of IREI and up regulating pJNK, which indicated a pro-apoptotic activity induced by IRE1 post irrecoverable ER stress This indicated that the ER stress induced apoptosis is triggered by LCL161, which might be important to overcome the ER induced protective effects induced by OBX.Conclusion: Taken together, our studies indicate that LCL161/OBX combination induces synergistic cell death through modulation of apoptosis, authophagy and the ER stress response. DisclosuresNo relevant conflicts of interest to declare.

Abstract 4178: Global assessment of the protein tyrosine phosphatome in cancer

Abstract Receptor tyrosine kinases and non-receptor tyrosine kinases are an important class of molecules controlling a large number of processes from growth to metabolic regulation. These tyrosine kinases have been well studied and scrutinized for their role in these normal processes as well as abnormal processes, such as cancer. Parallel to these kinases are more that 100 tyrosine phosphatases, which also fall into either receptor or non receptor classifications. While some of these phosphatases antagonize the tyrosine kinases others including many of the receptor bound phosphatases are critical in their own right in signaling processes such as pro survival, proliferation, etc. Although tyrosine kinases have been well studied, the study of tyrosine phosphatases has lagged. Numerous tyrosine phosphatases have been identified that are misregulated and important for cancer growth including PTPRD in breast cancer, and PTPRZ1 in small cell lung cancer as well as many others. However there has been no comprehensive study of all PTPs or the protein tyrosine phosphatome (PTPome), to identify which have a role in cancer. We as a cancer community are fortunate to have access to such a wide array of data types from a number of data sources such as TCGA, where we can determine if the PTPs are mis regulated in more than 20 types of cancers. We used TCGA data for all tumor types and subsetted the data to analyze the PTPome for all data types including expression, methylation, copy number and sequence data where available. Thus far we have identified known PTPs involved in a number of cancers, including PTPRD and PTPRZ1, but have also identified a number of PTPs altered in cancers that were less characterized such as the alteration of PTPRD in melanoma which is predominantly by mutations with much fewer deletions. While many of these mutations are missense and are difficult to characterize there are also a number of non-sense mutations, which would severely alter function as well as a number of splice site mutation of which some severely alter function and others are likely to alter function. The identification of this PTP is but one example to demonstrate the richness of this dataset. This analysis has been extremely useful to identify the PTPs that are altered in cancer and these results will be useful to many in the research community that are interested in PTP involvement in cancer but will also be of even greater use to those researchers who have not appreciated the vast importance of these under studied proteins. Citation Format: Nicholas Tonks, Robert Lucito. Global assessment of the protein tyrosine phosphatome in cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4178. doi:10.1158/1538-7445.AM2014-4178

Taxane-induced hedgehog signaling is linked to expansion of breast cancer stem-like populations after chemotherapy.

Recurrence of breast cancer after chemotherapy is thought to arise from resistant breast cancer stem cells which are eventually able to repopulate the tumor. The Hedgehog (HH) signaling pathway has been shown to regulate the proliferation and survival of breast cancer stem cells, and has been shown to promote resistance to chemotherapy through the activation of multi-drug resistance and pro survival pathways. Here we report that exposure of heterogenous breast cancer cell lines to docetaxel (DOC) resulted in release of Sonic Hedgehog ligand (SHH) and activation of the HH pathway as evidenced by increased expression and nuclear translocation of the downstream effector Gli-1 at 4-24 h after DOC treatment. This activation had little effect on the bulk of the tumor cell population as inhibition of HH signaling failed to increase apoptosis in response to DOC. However, HH pathway activation was required for clonogenic growth of cell lines after DOC. Increases in stemness markers as well as mammosphere formation were observed after treatment with DOC suggesting an increase in the breast cancer stem cell populations. These increases were similar to that of cell lines cultured in the presence of recombinant SHH and could be eliminated by co-treatment with HH inhibitors. These results suggest that HH pathway activation induced by DOC treatment does not have a chemosensitizing effect on the heterogeneous tumor population, but may be required for survival and expansion of breast cancer stem cells after chemotherapy.

Regulation of protumorigenic pathways by Insulin like growth factor binding protein2 and its association along with β-catenin in breast cancer lymph node metastasis

BackgroundInsulin like growth factor binding proteins modulate the mitogenic and pro survival effects of IGF. Elevated expression of IGFBP2 is associated with progression of tumors that include prostate, ovarian, glioma among others. Though implicated in the progression of breast cancer, the molecular mechanisms involved in IGFBP2 actions are not well defined. This study investigates the molecular targets and biological pathways targeted by IGFBP2 in breast cancer.MethodsTranscriptome analysis of breast tumor cells (BT474) with stable knockdown of IGFBP2 and breast tumors having differential expression of IGFBP2 by immunohistochemistry was performed using microarray. Differential gene expression was established using R-Bioconductor package. For validation, gene expression was determined by qPCR. Inhibitors of IGF1R and integrin pathway were utilized to study the mechanism of regulation of β-catenin. Immunohistochemical and immunocytochemical staining was performed on breast tumors and experimental cells, respectively for β-catenin and IGFBP2 expression.ResultsKnockdown of IGFBP2 resulted in differential expression of 2067 up regulated and 2002 down regulated genes in breast cancer cells. Down regulated genes principally belong to cell cycle, DNA replication, repair, p53 signaling, oxidative phosphorylation, Wnt signaling. Whole genome expression analysis of breast tumors with or without IGFBP2 expression indicated changes in genes belonging to Focal adhesion, Map kinase and Wnt signaling pathways. Interestingly, IGFBP2 knockdown clones showed reduced expression of β- catenin compared to control cells which was restored upon IGFBP2 re-expression. The regulation of β-catenin by IGFBP2 was found to be IGF1R and integrin pathway dependent. Furthermore, IGFBP2 and β-catenin are co-ordinately overexpressed in breast tumors and correlate with lymph node metastasis.ConclusionThis study highlights regulation of β-catenin by IGFBP2 in breast cancer cells and most importantly, combined expression of IGFBP2 and β-catenin is associated with lymph node metastasis of breast tumors.

Targeting the Cellular and Molecular Components of CD28 Mediated Survival Signaling in Multiple Myeloma

Abstract 722Very little is known about the cellular or molecular nature of pro-survival interactions between multiple myeloma (MM) and their bone marrow stromal hosts. CD28, the prototypical co-stimulatory molecule on T cells is overexpressed on MM relative to their normal counterparts. Clinical studies have independently shown that expression of CD28 or its ligand CD86 on myeloma cells portends poor survival in patients. We and others have shown that CD28 activation increases MM survival via NFkB accompanied by increased PI3K activity. Previously (ASH2010 and 2011), and since published, we have shown that myeloid dendritic cells (DC) form a vital part of the myeloma microenvironment, and myeloma cells interact with DC via a CD28-CD80/CD86 (B7) interaction. On the myeloma side, this interaction drives a CD28 mediated pro-survival signal, while on the DC side, it drives a B7 mediated induction of growth and immunosuppressive factors that modulate the MM survival niches in the bone marrow. In this work, we delineated key molecules downstream of CD28 that transduce the pro survival signal on the myeloma side as well as explored agents and strategies to target DC that help MM survive in the microenvironment. We also studied the role of DC IL-6 for MM survival in cocultures.We have previously shown that CD28 activation by anti-CD28 mAb involves activation of the multifunctional pro-survival transcription factor NFkB accompanied by increased PI3K activity. Through immunoassays and radioactivity assays, using myeloma cell lines that were transfected with wild type murine CD28 (mCD28 differs only by 3 amino acids from human hCD28) and mutants for PI3K binding site YMNM, we show that the CD28 ligation with anti-mCD28 antibodies induces immediate tyrosine phosphorylation of mCD28 (just as observed by others in T cells), followed by binding of p85, the catalytic subunit of PI3K and activation of VAV1 (Fig 1). Association of p85 and VAV1 activation depended on an intact PI3K binding site on CD28's cytoplasmic tail. Our studies on how this signal is transduced show no involvement of GRB2 or SLP76 more traditionally associated with CD28 signaling. Moreover, silencing SLP76 using siRNA did not affect antibody mediated protection of MM cells against serum starvation. Activation of human CD28 with anti-CD28 mAb also up-regulated the basal tyrosine phosphorylation of human CD28 further just as observed in murine CD28.Through EMSAs, we also show that similar to what we have previously seen with activating antibodies, increase in NFkB activity was observed in myeloma cells when they were cocultured with DCs while being exposed to melphalan, but this spike did not dissipate in the presence of B7 blocking reagents such as CTLA4Ig (Abatacept®) suggesting a low threshold of CD28 activation. Next we tried using agents that have been known to modulate microenvironment such as lenalidomide, a novel drug that is currently used in combination with other chemotherapeutic agents such as dexamethasone and Bortezomib (Velcade®). Survival assays using flow cytometry, show that lenalidomide treated DCs provided significantly lower protection against cell death compared to wild type DC. Lenalidomide DCs also show lower levels of B7 receptors which might be a contributing factor to this phenomenon. Blocking CD28-B7 interaction with CTLA4Ig or CD28(Fab) molecules reduced the pro-survival effect of DC on MM against dexamethasone (Fig 2) melphalan or bortezomib. In order to determine the role of IL-6 in an environment where myeloma cells derive pro-survival signals from their interaction with DC, we used neutralizing antibodies against human IL-6 in cocultures of MM cells with DC. Surprisingly, IL-6 neutralization did not affect the protective effect of DC on MM in cocultures in the presence of chemotherapeutic drugs like melphalan or dexamethasone (Fig 2).All these suggest that targeting DC might be a viable strategy towards augmenting chemotherapy, either by using agents that attenuate DC differentiation or agents that can block the CD28-B7 interaction between DC and MM. Moreover, MM cells do not seem to depend much on IL-6 as a survival factor when they are in contact with DC and its pro-survival effect is more visible when myeloma cells have to survive outside the microenvironment without DC (previous presentation at ASH 2011). [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Achyranthes aspera (Apamarg) leaf extract inhibits human pancreatic tumor growth in athymic mice by apoptosis

Ethnopharmacological relevanceAchyranthes aspera (Family Amaranthacea) is used for cancer therapy by ayurvedic medical practitioners in India. However, due to the non formal nature of its use, there are no systematic studies validating its medicinal properties. Thus, it's utility as an anti cancer agent remains anecdotal. Earlier, we demonstrated A. aspera to exhibit time and dose-dependent preferential cytotoxicity to cultured human pancreatic cancer cells. In this report we validate in vivo anti tumor properties of A. aspera. Materials and methodsThe in vivo anti tumor activity of leaf extract (LE) was tested by intraperitoneal (IP) injections into athymic mice harboring human pancreatic tumor subcutaneous xenograft. Toxicity was monitored by recording changes in behavioral, histological, hematological and body weight parameters. ResultsDosing LE to athymic mice by I.P. injection for 32 days showed no adverse reactions in treated mice. Compared to the control set, IP administration of LE to tumor bearing mice significantly reduced both tumor weight and volume. Gene expression analysis using Real time PCR methods revealed that LE significantly induced caspase-3 mRNA (p<0.001) and suppressed expression of the pro survival kinase Akt-1 (p<0.05). TUNEL assay and immunohistochemistry confirmed apoptosis induction by activation of caspase-3 and inhibiting Akt phosphorylation in treated sets. These results are in agreement with RT PCR data. ConclusionTaken together, these data suggest A. aspera to have potent anti cancer property.

Perifosine and capecitabine in metastatic colorectal cancer

Patients with metastatic colorectal cancer (mCRC) who were taking perifosine plus capecitabine had a median time to pro gression and survival of 27·5 weeks and 17·7 months, respectively, by contrast with 10·1 weeks and 7·6 months, respectively, for those taking capeci ta bine alone. Robert C Hermann (Northwest Georgia Oncology Centers, Marietta, GA, USA) said the results from this randomised, placebo-controlled phase 2 trial are encouraging, noting that patients with such advanced disease have very few drug options. A large phase 3 trial XPECT is underway with the combination treatment in refractory advanced colorectal cancer. Perifosine is an alkylphospholipid signal transduction modulator aff ecting PI3K/Akt/mTOR and JNK pathways, which promote cell proliferation and survival, apoptosis, and drug resistance. Perifosine might also aff ect the NF-kB pathway involved in cell proliferation and resistance in colorectal cancer. Tested alone, perifosine has not shown activity in mCRC. The trial, which included 38 patients, had an overall response rate of 20% in patients taking the two drugs, compared with 7% for monotherapy, and improvements were noted in a subset of fl uorouracil-refractory patients. Toxicities in the combination group included diarrhoea, fatigue, nausea, and hand–foot syndrome, and were higher than with capecitabine alone. Grade 3 to 4 adverse eff ects were mostly noted in patients taking the two drugs. By the trial’s conclusion, all patients had progressed: 18 in the combination group and all those taking capecitabine alone had died. Signifi cantly, nine fl uorouracilrefractory patients of 20 taking the combination had a partial response or stable disease compared with only three of 18 in the capecitabinealone group. Fluorouracil is related to capecitabine, so the results of this trial could mean that combining perifosine with capecitabine might ameliorate resistance to fl uorouracil. “While these results may be encouraging, they are preliminary,” said Leonard Saltz (Memorial SloanKettering Cancer Center, New York City, NY, USA). The trial was very small and the fi ndings must be confi rmed in a larger study, he said. It is also not clear yet what perifosine’s exact mechanism of action is, said Saltz. “Testing perifosine with capecitabine is like using a negative or placebo control, since capecitabine alone is not substantially active in fl uorouracil failures, and NCCN guidelines specifi cally note that it does not have substantial activity in 5FU failures,” Saltz said.

39 Endogenous protection against myocardial ischaemia-reperfusion injury in the diabetic heart

There are contradictory data in the literature regarding the susceptibility of the diabetic heart to ischaemia/reperfusion injury and the initiation of cardioprotection by activating endogenous prosurvival mechanisms. We hypothesise that...

Thyroid Hormone T3 Counteracts STZ Induced Diabetes in Mouse

This study intended to demonstrate that the thyroid hormone T3 counteracts the onset of a Streptozotocin (STZ) induced diabetes in wild type mice. To test our hypothesis diabetes has been induced in Balb/c male mice by multiple low dose Streptozotocin injection; and a group of mice was contemporaneously injected with T3. After 48 h mice were tested for glucose tolerance test, insulin serum levels and then sacrified. Whole pancreata were utilized for morphological and biochemical analyses, while protein extracts and RNA were utilized for expression analyses of specific molecules. The results showed that islets from T3 treated mice were comparable to age- and sex-matched control, untreated mice in number, shape, dimension, consistency, ultrastructure, insulin and glucagon levels, Tunel positivity and caspases activation, while all the cited parameters and molecules were altered by STZ alone. The T3-induced pro survival effect was associated with a strong increase in phosphorylated Akt. Moreover, T3 administration prevented the STZ-dependent alterations in glucose blood level, both during fasting and after glucose challenge, as well as in insulin serum level. In conclusion we demonstrated that T3 could act as a protective factor against STZ induced diabetes.

Highlights of This Issue

Although the role of HIF-1 in tumor progression is well characterized, the exact contribution of HIF-1 signaling to tumor radioresistance is not. Schwartz and colleagues leveraged high-resolution multimodality functional imaging of intact tumor xenografts to show that ischemic HIF-1 tumor signaling mediates downstream proangiogenic signaling responsible for the recovery of tumor blood supply following radiation treatment. Targeted HIF-1 blockade has the potential to provide clinically relevant radiosensitization through inhibition of a key mechanistic source for posttreatment stromal adaptation and tumor regrowth.TRAIL, a trigger of death receptor-induced apoptosis, is implicated in the clearance of disseminated tumor cells from the circulation. Despite promising results in animal models, current clinical work with TRAIL therapies suggests they require combination with sensitizing agents. The study by Phipps and coworkers shows that disruption of cell spreading can sensitize metastatic breast and melanoma cell lines to TRAIL-induced apoptosis in vitro. Disruption of signaling through β1 integrin, talin1, Src and MRTF-driven transcriptional control of the cytoskeleton all resulted in increased TRAIL killing. This work presents a range of targets that could be used to sensitize metastatic tumour cells to TRAIL therapies in vivo.According to previous studies, osteopontin (OPN) splicing isoforms show tumorspecific roles. To explore the role of OPN isoforms on ovarian carcinoma, Tilli and colleagues performed in vitro and in vivo functional assays using OvCaR-3 cell line. Among the 3 OPN isoforms, OPNc presented tumor-specific expression and specifically activated different features related to ovarian carcinoma tumor progression. OPNc features were mainly mediated by the PI3-K pro survival signaling, which is a crucial pathway in ovarian carcinoma progression. These results show promise for new therapeutic approaches for OC that selectively downregulate OPNc, altering its properties favoring ovarian carcinoma progression.Strategies to target functional activities of estrogen receptor (ER), prevalently abundant in the majority of breast cancers, fail in advanced stages of ER-independent, metastatic, drug-resistant cancer phenotypes. Sinha and coworkers developed the first estrogenic molecule (ESC8) that is nontoxic to normal cells while exhibiting anticancer activity in both ER(+/−) breast cancer cells. Mechanistically, ESC8 shows uncharacteristic PI3K/Aktassisted coinduction of apoptosis and autophagy in ER(−) breast cancer. Interfering with mTOR regulation, ESC8 induced autophagy for killing of cancer cells and prominently reduced mammary tumor growth. Together, ESC8 development strategized a novel concept of single drug chemotherapy for all stages of breast cancer.

TRAF6, a new member of the proximal signaling complex recruited by BAFFR and TACI in B lymphocytes (34.1)

Abstract The cytokines BAFF (B-cell activating factor of the TNF family, BLyS) and APRIL (a proliferation-inducing ligand) activate several major signaling cascades responsible for B-cell survival and homeostasis. Made primarily by myeloid cells, BAFF binds and activates three cell membrane receptors; BCMA (B-cell maturation antigen), TACI (transmembrane activator and CAML interactor), and BAFF-R (BAFF Receptor, BR3), while APRIL binds TACI and BCMA. Studies of genetically altered mice demonstrate that TACI and BCMA perform niche roles in B-cell Ig isotype switching and plasma cell maintenance. In contrast, BAFF-R deletion in mice results in greater than 90% loss of mature B-cells, revealing it as an essential mediator of B-cell maturation and survival beyond the immature transitional (T1) stage. TNF receptor family proteins utilize characteristic combinations of TNF receptor associated factors (TRAFs) to bridge receptor activation with a multitude of downstream signaling pathways. To date, studies of TRAF recruitment by TACI and BAFFR and their roles in signaling have been limited, and TRAF3 was the only TRAF thought to associate with these receptors. Here we present the novel finding that TRAF6 is directly recruited by TACI and BAFFR in B-cells, and that it forms an essential link to the initiation of the canonical NF-kB pathway and other pro survival signals.

Computational Modeling of Cell Survival/Death Using BiCMOS

A well-structured and controlled design methodology, along with a supporting hierarchical design system, has been developed to optimally support the development effort on several programs requiring gate array and semi custom Very Large Scale Integration (VLSI) design. In this paper, we will present an application of VLSI in System Biology. This work examines signaling networks that control the survival decision treated with combinations of three primary signals the pro death cytokine, tumor necrosis factor- α (TNF), and the pro survival growth factors, epidermal growth factor (EGF) and insulin. We have made model by taking three inputs, than made the truth table, Boolean equation and than implement the equation using BiCMOS logic circuit.

Expression and regulation of osteopontin in type 1 diabetes

Osteopontin (OPN) is a secreted acidic phosphoprotein that is involved in many inflammatory and immune-modulating disorders. We previously demonstrated that OPN is a novel islet protein and a pro survival factor that may serve as an intrinsic feedback regulator of nitric oxide signaling in β-cells. Here, we investigated the endogenous expression of pancreatic OPN in non obese diabetic (NOD) mice and explored its regulation in the islets and b-cells. High levels of pancreatic OPN mRNA and protein were seen in the prediabetic NOD mice pancreata. The temporal pattern of OPN expression inversely correlated with progression of insulitis and β-cell destruction. Immunostaining of pancreatic serial sections showed co localization of OPN with most of the islet hormones. Next we investigated the regulation of OPN in the islets and β-cells. Naturally occurring early upregulation of OPN transcription was seen after exposure of native normoglycemic NOD islets and β-cells to a high-dose combination of IL-1β, TNF-α and IFN-γ. To distinguish between the effect of cytokines and high glucose on OPN transcription, RINm5F cells were transfected with luciferase-labeled rat OPN promoter and treated with cytokines or glucose. Cytokines induced upregulation of OPN promoter activity within one hour, while glucose induced a dose-dependent upregulation of OPN promoter activity after 24 hrs. Long-term exposures to cytokines or glucose reduced OPN expression and promoter activity. Our data provide the first observations into the presence of a positive intrinsic mechanism that regulates pancreatic OPN expression. Based upon previous studies that support a protective role of OPN in the islets, our data suggest that exhaustion of this local OPN system is implicated in the associated loss of endogenous islet protection and progression of the destructive insulitis and diabetes severity in the NOD mouse model.