W1364 Protein Extracts from Hsd Smooth Muscle Allow Enteric Nerve Cell Growth and Survival In Vitro

Abstract Abstract 4965 The Phosphatidylinositol-3-kinase (PI3K)/AKT/mTOR pathway is frequently deregulated in Hodgkin (HL) and non-Hodgkin lymphoma (NHL), and has been linked with tumor cell growth and survival. Although several proteins/enzymes in this pathway can be targeted by a variety of small molecules in vitro and in vivo, it remains unclear which protein target is the ideal for clinical testing. Previous studies demonstrated that the clinical activity of mTOR inhibitors may be attenuated by a negative feedback loop that involves activation of AKT, suggesting that a dual inhibition of AKT and mTOR activation may produce a better therapeutic outcome. To test this hypothesis, we evaluated the in vitro activity of NVP-BEZ235, a dual inhibitor of PI3K and mTOR, in a panel of 13 HL and NHL cell lines. NVP-BEZ235 inhibited cell growth and induced apoptosis in lymphoma cell lines in a time and dose dependent manner. After 48 hours of incubation, the IC50 ranged between 50 and 100 nM, and it was equally effective in ABC and GCB-derived DLBCL cell lines. NVP-BEZ235 induced cell death was primarily due to induction of apoptosis, as evident by the annexin-V and PI dual staining method, and the induction of caspase 3 and PARP cleavage. NVP-BEZ235 effectively inhibited the activation of the PI3K pathway at several steps, including decreasing the phosphorylation level of p-Akt (Ser473), p-Akt (Thr308), p-mTOR, p-4-EBPI and pP70S6K. Because lymphoma cells frequently depend on multiple activated signaling pathways to promote their survival, including the JAK/STAT pathway, we investigated the potential synergy between PI3K and JAK/STAT pathway inhibitors. Lymphoma cells were variably sensitive to the JAK1/2 inhibitor INCB16562 in vitro. Submaximal concentrations of NVP-BEZ235 demonstrated a synergistic activity with INCB16562. Collectively, our data show that the PI3K/mTOR inhibitor NVP-BEZ235 is highly effective against a wide range of lymphoma cell lines, and warrants evaluating it alone and in combination with JAK/STAT inhibitors in phase I/II clinical trials in patients with relapsed lymphoma. Disclosures: No relevant conflicts of interest to declare.

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Genistein inhibits tumour cell growth in vitro but enhances mitochondrial reduction of tetrazolium salts: A further pitfall in the use of the MTT assay for evaluating cell growth and survival

European Journal of Cancer ◽

10.1016/0959-8049(93)90297-s ◽

1993 ◽

Vol 29 (11) ◽

pp. 1573-1577 ◽

Cited By ~ 94

Author(s):

M.C. Pagliacci ◽

F. Spinozzi ◽

G. Migliorati ◽

G. Fumi ◽

M. Smacchia ◽

...

Keyword(s):

Cell Growth ◽

Mtt Assay ◽

Tumour Cell ◽

Growth And Survival ◽

Tetrazolium Salts ◽

Tumour Cell Growth ◽

Cell Growth And Survival

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Human Monoclonal Antibody Targeting IL-17A (AIN457) Down-Regulates MM Cell-Growth and Survival and Inhibits Osteoclast Development In Vitro and In Vivo: A Potential Novel Therapeutic Application In Myeloma

Blood ◽

10.1182/blood.v116.21.456.456 ◽

2010 ◽

Vol 116 (21) ◽

pp. 456-456

Author(s):

Rao H Prabhala ◽

Mariateresa Fulciniti ◽

Dheeraj Pelluru ◽

Puru Nanjappa ◽

Christine Pai ◽

...

Keyword(s):

Monoclonal Antibody ◽

Cell Growth ◽

Stromal Cells ◽

Human Monoclonal Antibody ◽

Critical Role ◽

Osteoclast Differentiation ◽

Growth And Survival ◽

Cell Growth And Survival

Abstract Abstract 456 We have previously demonstrated that IL-17 producing TH17cells, a new subset of T helper cells, are significantly elevated in peripheral blood and bone marrow (BM) from patients with multiple myeloma (MM) and IL-17 produced by these cells promotes MM cell growth and survival, suppresses immune responses and induces osteoclast differentiation both in vitro and in vivo. Based on these observations we have investigated the effects of human anti-IL-17A monoclonal antibody (mAb), AIN-457, in MM. We observed that whereas IL-17A induced proliferation of MM cells (+30.7+2.7%) compared to control; anti-IL-17A mAb AIN-457 significantly inhibited MM cell growth both in presence and absence of BM stromal cells, as measured by thymidine incorporation (−18.7+1.5% and −22.7+2.6% respectively). We have further confirmed these inhibitory effects of anti-IL-17A antibody using MM cell colony forming assay with MethoCult agar plates. While presence of IL-17A increased the colony number from 80 in control plates to 188, presence of AIN-457 reduced the colonies to <40 per unit area (p < 0.01). Evaluating the mechanism of action, IL-17A induced IL-6 production (+289.6+38%; p<0.01); while AIN457 significantly down-regulated IL-6 production (−25+7%; p<0.05) in MM-BMSC co-culture. We also observed that AIN-457 significantly reduced adhesion of MM cells to stromal cells (27%, p=0.011). AIN457 significantly inhibited IL-6 production in human fetal bone chips in the presence of MM cells within 24 hours of ex-vivo culture (control − 487+39 pg/ml; IL-17 990+27 pg/ml; p<0.01 and AIN-457 − 326+7 pg/ml; p<0.01). Since IL-17A plays a critical role in bone damage, we further evaluated the effect of this mAb on the generation of osteoclasts. When normal BM cells were cultured for three weeks in osteoclast supporting medium, presence of AIN-457 significantly inhibited TRAP+ multinucleated osteoclast cell numbers by>60%. We next evaluated the efficacy of AIN-457 in vivo in the murine models of human myeloma; in the subcutaneous MM xenograft model, we observed significant reduction in tumor volumes by pre-treatment with AIN457 compared to control (142+77 mm versus 355+56 mm, p=0.019) while IL-17A significantly increased MM cell growth (727+135 mm, p=0.01). More importantly in the SCIDhu model of human myeloma where MM cells grow within the human microenvironment in the mice, administration of AIN-457 weekly for 4 weeks after the first detection of tumor in mice led to a significant inhibition of tumor growth as measured by human sIL-6 receptor compared to control mice (5.9±2.2 ng/ml versus 23.2±6.3 ng/ml; n=7; P <0.01). These pre-clinical in vitro and in vivo observations confirm the role of IL-17A produced by TH17 cells in MM and provide the rationale for clinical evaluation of AIN 457 for both anti-myeloma effects as well as to improve bone disease in myeloma. Disclosures: No relevant conflicts of interest to declare.

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Multiple signaling pathways promote B lymphocyte stimulator–dependent B-cell growth and survival

Blood ◽

10.1182/blood-2007-03-077222 ◽

2008 ◽

Vol 111 (2) ◽

pp. 750-760 ◽

Cited By ~ 128

Author(s):

Robert T. Woodland ◽

Casey J. Fox ◽

Madelyn R. Schmidt ◽

Peter S. Hammerman ◽

Joseph T. Opferman ◽

...

Keyword(s):

B Cells ◽

Cell Growth ◽

B Cell ◽

Signaling Pathways ◽

B Lymphocyte ◽

Marginal Zone B Cells ◽

Growth And Survival ◽

B Lymphocyte Stimulator ◽

Cell Growth And Survival

We investigated the mechanism by which B lymphocyte stimulator (BLyS)/BAFF, a tumor necrosis factor superfamily ligand, promotes B-cell survival and resistance to atrophy. BLyS stimulation activates 2 independent signaling pathways, Akt/mTOR and Pim 2, associated with cell growth and survival. BLyS blocks the cell volume loss (atrophy) that freshly isolated B cells normally undergo when maintained in vitro while concurrently increasing glycolytic activity and overall metabolism. This atrophy resistance requires Akt/mTOR. We used a genetic approach to resolve the contributions of Akt/mTOR and Pim kinase pathways to BLyS-mediated survival. Pim 2–deficient B cells are readily protected from death by BLyS stimulation, but this protection is completely abrogated by treatment with the mTOR inhibitor rapamycin. Furthermore, rapamycin treatment in vivo significantly reduces both follicular and marginal zone B cells in Pim-deficient but not healthy hosts. BLyS-dependent survival requires the antiapoptotic protein Mcl-1. Mcl-1 protein levels rise and fall in response to BLyS addition and withdrawal, respectively, and conditional deletion of the Mcl-1 gene renders B cells refractory to BLyS-mediated protection. Because BlyS is required for the normal homeostasis of all B cells, these data suggest a therapeutic strategy simultaneously inhibiting mTOR and Pim 2 could target pathogenic B cells.

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