Cecropin Suppresses Human Hepatocellular Carcinoma BEL-7402 Cell Growth and Survival in vivo without Side-Toxicity

Abstract P21-activated serine/threonine kinase 4 (PAK4) is a major effector of Cdc42 and is essential for cytoskeleton reorganization. PAK4 is activated in cancer cells, promotes cell migration and anchorage-independent cell growth, and protects against apoptosis induction. With cellular migration playing a significant role in multiple myeloma (MM) cell growth and survival, we investigated the expression and subcellular localization of PAK4 in MM cells. We observed a high level of un-phosphorylated PAK4 in the cytoplasm and high levels of phosphorylated PAK4 in the nucleus. In a gain-of-function study, over-expression of PAK4-eGFP in PAK4-deficient MM cells (RPMI8226) significantly increased cell proliferation and survival. Conversely, in a loss-of-function study, conditional knock-down of PAK4 expression with TRIPZ-lentiviral vectors decreased MM cell proliferation and survival proportionally to the reduction in PAK4. With a significant impact of PAK4 on MM cell growth, we identified a class of orally bioavailable PAK4 allosteric modulators (PAMs; e.g. KPT-6604, -7189, -7657, -8752). We observed inhibition of MM cell growth and survival after treatment with PAMs even in the presence of bone marrow microenvironment. In addition, there is a significant correlation between PAK4 expression and the inhibition concentration (IC50s) of PAMs in proliferation assays. Moreover, inhibition of PAK4 induced receptor and mitochondrial-mediated apoptotic pathways via Caspase-3, -8, and -9 activation. PAMs had no significant effect on normal PBMCs, suggesting a favorable therapeutic index in MM treatment. Finally, in two murine models of human myeloma, orally bioavailable KPT-8752 given daily was able to inhibit tumor growth in vivo and prolong overall survival. In summary, PAK4 plays an important cellular and molecular function in myeloma and its inhibition with a new class of PAK4 allosteric modulators provides a novel therapeutic approach for the treatment of MM. Disclosures Senapedis: Karyopharm: Employment. Baloglu:Karyopharm: Employment. Anderson:Celgene: Consultancy; Sanofi-Aventis: Consultancy; Onyx: Consultancy; Acetylon: Scientific Founder, Scientific Founder Other; Oncoprep: Scientific Founder Other; Gilead Sciences: Consultancy.

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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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Brain-Derived Neurotrophic Factor Promotes Implantation and Subsequent Placental Development by Stimulating Trophoblast Cell Growth and Survival

Endocrinology ◽

10.1210/en.2009-0213 ◽

2009 ◽

Vol 150 (8) ◽

pp. 3774-3782 ◽

Cited By ~ 83

Author(s):

Kazuhiro Kawamura ◽

Nanami Kawamura ◽

Wataru Sato ◽

Jun Fukuda ◽

Jin Kumagai ◽

...

Keyword(s):

Cell Growth ◽

Neurotrophic Factor ◽

Brain Derived Neurotrophic Factor ◽

Trophoblast Cell ◽

Phosphatidylinositol 3 Kinase ◽

Trophoblast Cells ◽

Placental Development ◽

Growth And Survival ◽

Cell Growth And Survival

Successful implantation of the blastocyst and subsequent placental development is essential for reproduction. Expression of brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5, together with their receptor, tyrosine kinase B (TrkB), in trophectoderm cells of blastocyst suggests their potential roles in implantation and placental development. Here we demonstrated that treatment with BDNF promoted blastocyst outgrowth, but not adhesion, in vitro and increased levels of the cell invasion marker matrix metalloproteinase-9 in cultured blastocysts through the phosphatidylinositol 3-kinase pathway. After implantation, BDNF and neurotrophin-4/5 proteins as well as TrkB were expressed in trophoblast cells and placentas during different stages of pregnancy. Both TrkB and its ligands were also expressed in decidual cells. Treatment of cultured trophoblast cells with the TrkB ectodomain, or a Trk receptor inhibitor K252a, suppressed cell growth as reflected by decreased proliferation and increased apoptosis, whereas an inactive plasma membrane nonpermeable K252b was ineffective. Studies using the specific inhibitors also indicated the importance of the phosphatidylinositol 3-kinase/Akt pathway in mediating the action of TrkB ligands. In vivo studies in pregnant mice further demonstrated that treatment with K252a, but not K252b, suppressed placental development accompanied by increases in trophoblast cell apoptosis and decreases in placental labyrinth zone at midgestation. In vivo K252a treatment also decreased fetal weight at late gestational stages. Our findings suggested important autocrine/paracrine roles of the BDNF/TrkB signaling system during implantation, subsequent placental development, and fetal growth by increasing trophoblast cell growth and survival.

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