scholarly journals Cytotoxic Effect of Icaritin and Its Mechanisms in Inducing Apoptosis in Human Burkitt Lymphoma Cell Line

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Zi-Jian Li ◽  
Can Yao ◽  
Su-Fang Liu ◽  
Long Chen ◽  
Ya-Ming Xi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Burkitt Lymphoma ◽  
Cell Apoptosis ◽  
Potential Effect ◽  
Signal Molecules ◽  
Antitumor Activities ◽  
Caspase 9 ◽  
Human Solid Tumor ◽  
Burkitt Lymphoma Cell Line ◽  
Induced Apoptosis

Icaritin (ICT), a hydrolytic product of icariin fromEpimedium genus, exhibits antitumor activities in several human solid-tumor and myeloid leukemia cells with extensive influence on various cell signal molecules, such as MAPKs being involved in cell proliferation and Bcl-2 participating in cell apoptosis. However, the effect of icaritin on Burkitt Lymphoma has not been elucidated. In the present study, we first screened the potential effect of icaritin on Burkitt lymphoma Raji and P3HR-1 cell lines and found that icaritin showed cytotoxicity in both cell lines. We further found that icaritin could significantly inhibit Raji cells proliferation with S-phase arrest of cell cycle and induced cell apoptosis accompanied by activation of caspase-8 and caspase-9 and cleavage of PARP. We also observed that icaritin was able to decrease Bcl-2 levels, thus shifting the Bcl-2/Bax ratio, and it could obviously reduce c-Myc, a specific molecular target in Burkitt lymphoma. Our findings demonstrated that icaritin showed cytotoxicity, inhibited cell growth, caused S arrest, and induced apoptosis in Burkitt lymphoma cells and provided a rationale for the further evaluation of icaritin for Burkitt lymphoma therapy.

scholarly journals Ailanthone Promotes Human Vestibular Schwannoma Cell Apoptosis and Autophagy by Downregulation of miR-21

2018 ◽  
Vol 26 (6) ◽  
pp. 941-948 ◽  
Author(s):  
Peizhen Yang ◽  
Dezhong Sun ◽  
Fei Jiang
Keyword(s):  
Cell Proliferation ◽  
Cell Apoptosis ◽  
Caspase 3 ◽  
Apoptotic Cell ◽  
Ailanthus Altissima ◽  
Antitumor Activities ◽  
Caspase 9 ◽  
Chinese Medicinal Herb ◽  
Induced Apoptosis ◽  
Functional Actions

Ailanthone (AIL) is a quassinoid isolated from the traditional Chinese medicinal herb Ailanthus altissima. The antitumor activities of AIL have been reported in several cancers. The purpose of the present study was to explore the effect of AIL on vestibular schwannomas (VSs). Various concentrations of AIL (0‐1 μM) were used to treat human primary VS cells, and then cell viability, proliferation, apoptosis, and autophagy were assessed. Expression of miR-21 in VS cells was altered by miRNA transfection. The functional actions of AIL on miR-21 dysregulated cells were also assessed. AIL significantly reduced the viability of VS cells, and the IC50 value was 0.48 ± 0.023 μM. In response to 0.6 μM AIL, BrdU+ cell rate and cyclin D1 expression were reduced, apoptotic cell rate was increased, caspase 3 and caspase 9 were cleaved, Beclin-1 and LC3-II were accumulated, and p62 was downregulated. miR-21 was lowly expressed in AIL-treated cells, and AIL-induced apoptosis and autophagy were attenuated by miR-21 overexpression. In addition, AIL downregulated Ras and Raf and deactivated MEK, ERK, mTOR, and p70S6K, while the downregulation and deactivation induced by AIL were reversed by miR-21 overexpression. To conclude, AIL inhibited VS cell proliferation and induced apoptosis and autophagy. The antitumor activities of AIL in VS cells were realized possibly via downregulation of miR-21 and blocking the Ras/Raf/MEK/ERK and mTOR pathways.

Differential activation of ER stress and apoptosis in response to chronically elevated free fatty acids in pancreatic β-cells

2008 ◽  
Vol 294 (3) ◽  
pp. E540-E550 ◽  
Author(s):  
Elida Lai ◽  
George Bikopoulos ◽  
Michael B. Wheeler ◽  
Maria Rozakis-Adcock ◽  
Allen Volchuk
Keyword(s):  
Er Stress ◽  
Cell Lines ◽  
Cell Apoptosis ◽  
Human Islets ◽  
Pancreatic Β Cell ◽  
Β Cell ◽  
Min6 Cells ◽  
Relative Contribution ◽  
Induced Apoptosis

Chronic exposure to elevated saturated free fatty acid (FFA) levels has been shown to induce endoplasmic reticulum (ER) stress that may contribute to promoting pancreatic β-cell apoptosis. Here, we compared the effects of FFAs on apoptosis and ER stress in human islets and two pancreatic β-cell lines, rat INS-1 and mouse MIN6 cells. Isolated human islets cultured in vitro underwent apoptosis, and markers of ER stress pathways were elevated by chronic palmitate exposure. Palmitate also induced apoptosis in MIN6 and INS-1 cells, although the former were more resistant to both apoptosis and ER stress. MIN6 cells were found to express significantly higher levels of ER chaperone proteins than INS-1 cells, which likely accounts for the ER stress resistance. We attempted to determine the relative contribution that ER stress plays in palmitate-induced β-cell apoptosis. Although overexpressing GRP78 in INS-1 cells partially reduced susceptibility to thapsigargin, this failed to reduce palmitate-induced ER stress or apoptosis. In INS-1 cells, palmitate induced apoptosis at concentrations that did not result in significant ER stress. Finally, MIN6 cells depleted of GRP78 were more susceptible to tunicamycin-induced apoptosis but not to palmitate-induced apoptosis compared with control cells. These results suggest that ER stress is likely not the main mechanism involved in palmitate-induced apoptosis in β-cell lines. Human islets and MIN6 cells were found to express high levels of stearoyl-CoA desaturase-1 compared with INS-1 cells, which may account for the decreased susceptibility of these cells to the cytotoxic effects of palmitate.

scholarly journals Dihydrochalcone Derivative Induces Breast Cancer Cell Apoptosis via Intrinsic, Extrinsic, and ER Stress Pathways but Abolishes EGFR/MAPK Pathway

2019 ◽  
Vol 2019 ◽  
pp. 1-18 ◽  
Author(s):  
Wasitta Rachakhom ◽  
Patompong Khaw-on ◽  
Wilart Pompimon ◽  
Ratana Banjerdpongchai
Keyword(s):  
Breast Cancer ◽  
Er Stress ◽  
Cell Lines ◽  
Cell Apoptosis ◽  
Mapk Pathway ◽  
Annexin V ◽  
Mapk Signaling ◽  
Dependent Manner ◽  
Induced Apoptosis ◽  
Mcf 7

Dihydrochalcone derivatives are active compounds that have been purified from the Thai medicinal plant Cyathostemma argenteum. The objectives of this study were to investigate the effects of two dihydrochalcone derivatives on human breast cancer MDA-MB-231 and MCF-7 cell proliferation and to study the relevant mechanisms involved. The two dihydrochalcone derivatives are 4′,6′-dihydroxy-2′,4-dimethoxy-5′-(2″-hydroxybenzyl)dihydrochalcone (compound 1) and calomelanone (2′,6′-dihydroxy-4,4′-dimethoxydihydrochalcone, compound 2), both of which induced cytotoxicity toward both cell lines in a dose-dependent manner by using MTT assay. Treatment with both derivatives induced apoptosis as determined by annexin V-FITC/propidium iodide employing flow cytometry. The reduction of mitochondrial transmembrane potential (staining with 3,3′-dihexyloxacarbocyanine iodide, DiOC6, employing a flow cytometer) was established in the compound 1-treated cells. Compound 1 induced caspase-3, caspase-8, and caspase-9 activities in both cell lines, as has been determined by specific colorimetric substrates and a spectrophotometric microplate reader which indicated the involvement of both the extrinsic and intrinsic pathways. Calcium ion levels in mitochondrial and cytosolic compartments increased in compound 1-treated cells as detected by Rhod-2AM and Fluo-3AM intensity, respectively, indicating the involvement of the endoplasmic reticulum (ER) stress pathway. Compound 1 induced cell cycle arrest via enhanced atm and atr expressions and by upregulating proapoptotic proteins, namely, Bim, Bad, and tBid. Moreover, compound 1 significantly inhibited the EGFR/MAPK signaling pathway. In conclusion, compound 1 induced MDA-MB-231 and MCF-7 cell apoptosis via intrinsic, extrinsic, and ER stress pathways, whereas it ameliorated the EGFR/MAPK pathway in the MCF-7 cell line. Consequently, it is believed that compound 1 could be effectively developed for cancer treatments.

scholarly journals Topical 2′-Hydroxyflavanone for Cutaneous Melanoma

Cancers ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 1556 ◽  
Author(s):  
Bose ◽  
Singh ◽  
Igid ◽  
Green ◽  
Singhal ◽  
...  
Keyword(s):  
Cell Lines ◽  
Topical Therapy ◽  
Systemic Absorption ◽  
Caspase 9 ◽  
Epidermal Growth ◽  
Dietary Flavonoid ◽  
Parp1 Inhibitors ◽  
Induced Apoptosis ◽  
Immunocompetent Mice

2′-hydroxyflavanone (2HF) is a dietary flavonoid with anticancer activity towardsmultiple cancers. Here, we report that topically applied 2HF inhibits the growth of intradermalimplants of melanoma in immunocompetent mice. 2HF induced apoptosis and inhibited the growthof the human SK-MEL-24 as well as murine B16-F0 and B16-F10 melanoma cell lines in vitro.Apoptosis was associated with depletion of caspase-3, caspase-9, and PARP1 in B16-F0 and SKMEL-24 cells. Caspase-9 and MEKK-15 were undetected even in untreated B16-F10 cells. Signalingproteins TNFα, and phospho-PDGFR-β were depleted in all three cell lines; MEKK-15 was depletedby 2HF in SK-MEL-24 cells. 2HF enhanced sunitinib (an MEK and PDGFR-β inhibitor) and AZD2461 (a PARP1 inhibitor) cytotoxicity. 2HF also depleted the Ral-regulated, stress-responsive,antiapoptotic endocytic protein RLIP76 (RALBP1), the inhibition of which has previously beenshown to inhibit B16-F0 melanoma growth in vivo. Functional inhibition of RLIP76 was evidentfrom inhibition of epidermal growth factor (EGF) endocytosis by 2HF. We found that topicallyapplied 2HF–Pluronic Lecithin Organogel (PLO) gel inhibited B16-F0 and B16-F10 tumorsimplanted in mice and caused no overt toxicity despite significant systemic absorption. 2HFtreatment reduced phospho-AKT, vimentin, fibronectin, CDK4, cyclinB1, and BCL2, whereas itincreased BIM and phospho-AMPK in excised tumors. Several cancer signals are controlled byendocytosis, a process strongly inhibited by RLIP76 depletion. We conclude that 2HF–PLO gel maybe useful for topical therapy of cutaneous metastases of melanoma and could enhance theantineoplastic effects of sunitinib and PARP1 inhibitors. The mechanism of action of 2HF inmelanoma overlaps with RLI76 inhibitors.

scholarly journals Rana catesbeiana ribonuclease induces cell apoptosis via the caspase-9/-3 signaling pathway in human glioblastoma DBTRG, GBM8901 and GBM8401 cell lines

2015 ◽  
Vol 9 (6) ◽  
pp. 2471-2476 ◽  
Author(s):  
JEN-NI CHEN ◽  
GIOU-TENG YIANG ◽  
YI-FAN LIN ◽  
PEI-LUN CHOU ◽  
TSAI-KUN WU ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Cell Lines ◽  
Cell Apoptosis ◽  
Rana Catesbeiana ◽  
Caspase 9 ◽  
Human Glioblastoma

scholarly journals Synthesis and biological evaluation of novel benzo[b]xanthone derivatives as potential antitumor agents

2013 ◽  
Vol 78 (9) ◽  
pp. 1301-1308 ◽  
Author(s):  
Lin Luo ◽  
Jiang-Ke Qin ◽  
Zhi-Kai Dai ◽  
Shi-Hua Gao
Keyword(s):  
Cell Lines ◽  
Antitumor Agents ◽  
Biological Evaluation ◽  
Structural Factors ◽  
Anticancer Activities ◽  
Antitumor Activities ◽  
Mtt Method ◽  
Human Solid Tumor ◽  
Synthesis And Biological Evaluation

Nine novel aminoalkoxy substituted benzoxanthones (3a-3i) were synthesized. Their antitumor activities were evaluated in five human solid tumor cell lines including Hep-G2, BEL-7402, HeLa, MGC-803 and CNE by MTT method. The results showed that most of the compounds displayed moderate to good inhibitory activities on the tested cancer cell lines in vitro, among them compounds 3a and 3h showed higher antitumor activity than other tested compounds against most cell lines. The influence of two kinds of structural factors including the terminal amino group and length of carbon spacers on the anticancer activities were explored to discuss the preliminary structure-activity relationships.

Novel targets for endoplasmic reticulum stress-induced apoptosis in B-CLL

Blood ◽  
2010 ◽  
Vol 116 (15) ◽  
pp. 2713-2723 ◽  
Author(s):  
Emanuela Rosati ◽  
Rita Sabatini ◽  
Giuliana Rampino ◽  
Filomena De Falco ◽  
Mauro Di Ianni ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Cell Apoptosis ◽  
Ex Vivo ◽  
Lymphocytic Leukemia ◽  
Minor Role ◽  
Caspase 8 ◽  
Caspase 9 ◽  
Cytochrome C Release ◽  
Induced Apoptosis

Abstract A better understanding of apoptotic signaling in B-chronic lymphocytic leukemia (B-CLL) cells may help to define new therapeutic strategies. This study investigated endoplasmic reticulum (ER) stress signaling in spontaneous apoptosis of B-CLL cells and whether manipulating ER stress increases their apoptosis. Results show that a novel ER stress-triggered caspase cascade, initiated by caspase-4 and involving caspase-8 and -3, plays an important role in spontaneous B-CLL cell apoptosis. ER stress-induced apoptosis in B-CLL cells also involves CHOP/GADD153 up-regulation, increased JNK1/2 phosphorylation, and caspase-8–mediated cleavage of Bap31 to Bap20, known to propagate apoptotic signals from ER to mitochondria. In ex vivo B-CLL cells, some apoptotic events associated with mitochondrial pathway also occur, including mitochondrial cytochrome c release and caspase-9 processing. However, pharmacologic inhibition studies show that caspase-9 plays a minor role in B-CLL cell apoptosis. ER stress also triggers survival signals in B-CLL cells by increasing BiP/GRP78 expression. Manipulating ER signaling by siRNA down-regulation of BiP/GRP78 or treating B-CLL cells with 2 well-known ER stress-inducers, tunicamycin and thapsigargin, increases their apoptosis. Overall, our findings show that ER triggers an essential pathway for B-CLL cell apoptosis and suggest that genetic and pharmacologic manipulation of ER signaling could represent an important therapeutic strategy.

Fenretinide (4HPR) Inhibits Growth of Myeloma Cells in Their Microenvironment and Is a Potent Inhibitor of Angiogenesis and Osteoclastogenesis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3480-3480
Author(s):  
Xin Li ◽  
Wen Ling ◽  
Rinku Saha ◽  
Paul Perkins ◽  
Angela Pennisi ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Cell Lines ◽  
Mtt Assay ◽  
Cell Apoptosis ◽  
Mononuclear Cells ◽  
Annexin V ◽  
Dependent Manner ◽  
Angiogenic Potential ◽  
Inhibition Of Angiogenesis ◽  
Induced Apoptosis

Abstract Fenretinide (4HPR) is a relatively safe neoclassical retinoid analog that inhibits growth of various tumors through increased intracellular ceramide and ROS, induction of tumor cell apoptosis and inhibition of angiogenesis. 4HPR has been successfully tested as a chemopreventive and chemotherapeutic agent in clinical trials on various malignancies. In contrast to retinoic acid, 4HPR induces cell apoptosis rather than differentiation and shows synergistic responses with chemotherapeutic drugs in different tumor cell types. The biological effect and therapeutic value in multiple myeloma (MM) has not been investigated. The aim of this study was to investigate the anti-MM effect and mechanism of action of 4HPR using 3 stroma-dependent and 2 stroma-independent MM cell lines established in our laboratory, CD138-selected primary MM cells and co-culture systems of these cells with human osteoclasts and mesenchymal stem cells (MSCs) as previously described (Yaccoby et al., Cancer Res 2004). MM cell apoptosis detected by annexin V flow cytometry and TUNNEL, tumor growth by MTT assay, changes in caspase 3, 8 and 9 activity using Western blotting and ROS production by 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) dye assay. 4HPR inhibits growth of all tested MM cells in a dose- and time-dependent manner. The IC50 after 48 hrs in serum-containing media was 10 μM using MTT assay. 4HPR (3 μM) increased percent of apoptotic MM cells by 2.5±0.4 folds (p<0.01). Co-culture of these cell lines with osteoclasts only partially protected MM cells from the proapoptotic effect of this drug. Furthermore, 4HPR also induced apoptosis of primary CD138-selected MM cells co-cultured with osteoclasts or MSCs, and inhibited growth of bortezomib-resistant MM cell lines. In contrast, 4HPR had only minimal cytotoxic effect on blood mononuclear cells and MSCs. The proapoptotic effect of 4HPR involved increased level of ROS by 2.55±0.67 folds in MM cells (p<0.01). We also detected reduced levels of procaspase and increased cleaved caspase 8, 9 and 3 within 24 hrs of incubation with this drug. Sphingosine-1 phosphate (S1P) partially protected MM cells from 4HPR-induced apoptosis suggesting that, as reported for other tumors, anti-MM mechanism of this drug involved increased intracellular ceramide. 4HPR significantly inhibited tube formation by HUVEC in a matrigel assay (p<0.0001), confirming its anti-angiogenic potential. This drug also effectively prevented formation of multinucleated osteoclasts in culture of human osteoclast precursors with RANKL and M-CSF (p<0.0001). Furthermore, mature osteoclasts viability as assessed by MTT assays was reduced following incubation with 3 μM 4HPR (p<0.0001). We conclude that 4HPR is a potent anti-MM agent, affecting growth of MM cells in their microenvironment directly through induction of apoptosis in mechanisms involving ROS, caspase and possibly ceramide, and indirectly through inhibition of angiogenesis and osteoclastogenesis. Our data also suggests that S1P, which is highly produced by activated platelets, is an important survival factor for MM cells. Study is underway to test anti-MM efficacy of 4HPR in the SCID-hu model for primary myeloma.

The Tak-1 Inhibitor AZ-Tak1 Inhibits XIAP, Activates Caspase-9, and Induces Apoptosis in Mantle Cell Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1692-1692 ◽  
Author(s):  
Daniela Buglio ◽  
Sangeetha Palakurthi ◽  
Katharine F. Byth ◽  
Anas Younes
Keyword(s):  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Small Molecule ◽  
Cell Lymphoma ◽  
Small Molecule Inhibitor ◽  
Caspase 9 ◽  
Lymphoma Cells ◽  
Molecule Inhibitor ◽  
Mantle Cell ◽  
Induced Apoptosis

Abstract Abstract 1692 Poster Board I-718 Transforming growth factor-b-activated kinase 1 (TAK1) is a key regulator of NF-kB activation. TAK1 can be activated by a variety of pro-inflammatory cytokines and T and B cell receptors. Recent experiments demonstrated that deletion of TAK1 results in inactivation of both JNK and NF-kB signaling resulting in massive apoptotic death of hematopoietic cells in mice. In this study, we examined the expression pattern of TAK1 and its role as a potential therapeutic target for lymphoma. First, we examined TAK1 expression in a panel of lymphoid cell lines by western blot, and found it to be highly expressed in mantle cell lymphoma cell lines (Mino, SP53, and Jeko-1). These lines expressed relatively low levels of the tumor suppressor protein A20. Mino and SP53 expressed high level of p-p38. Subsequently, we investigated the in vitro activity of the novel TAK1 small molecule inhibitor AZ-Tak1 in these cell lines. AZ-Tak1 is a potent and a relatively selective inhibitor of TAK1 kinase activity, with an IC50 of 0.009 mM. It also inhibits Jak2 but at a much higher concentration (IC50=0.18 mM). AZ-Tak1 treatment decreased the level of p38 and ERK in mantle cell lymphoma cells, and induced apoptosis in a dose and time dependent manner, with an IC50 of 0.1-0.5 mM. Using the annexin-V and PI staining and FACS analysis, After 48 hours of incubation, AZ-Tak1 (0.1 mM) induced apoptosis in 28%, 34% and 86% of Mino, SP53, and Jeko cells, respectively, which was increased to 32%, 42%, and 86% when 0.5 mM concentration was used. Similar activity was also observed when primary mantle cell lymphoma cells were examined. Using pathway-specific protein arrays focusing on apoptosis, kinases, and transcription factors, AZ-Tak1 (0.5 mM) altered the level of several proteins that regulate cell growth and survival, especially members of the inhibitors of apoptosis (IAP) family. Specifically, AZ-Tak1 decreased the level of SMAC/DIABOLO and cytochrome –C in the mitochondria, which was associated with a decrease in the level of the anti-apoptotic protein X-linked IAP (XIAP) and activation of the intrinsic apoptotic pathway as evident by activation of caspase 9, cleavage of caspase 3, and induction of apoptosis. Furthermore, and consistant with its ability to inhibit Jak2 activity, AZ-Tak1 reduced STAT2 and STAT6 levels. AZ-Tak1 demonstrated no significant effect on bcl-2 family members. Finally, co-treatment with HDAC inhibitors demonstrated synergistic effect with low concentrations of AZ-Tak1. Collectively, our data demonstrate that targeting TAK1 by the small molecule inhibitor AZ-Tak1 induces cell death in mantle cell lymphoma by activating the intrinsic apoptosis pathway, suggesting that targeting TAK1 may have a therapeutic value for the treatment of mantle cell lymphoma. Disclosures Palakurthi: Astra Zeneca: Employment. Byth:Astra Zeneca : Employment.

NPI-2358, a Novel Vascular Disrupting Agent Blocks Growth and Angiogenesis in Multiple Myeloma Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3842-3842
Author(s):  
Dharminder Chauhan ◽  
Ajita V. Singh ◽  
Madhavi Bandi ◽  
Noopur Raje ◽  
Robert L Schlossman ◽  
...  
Keyword(s):  
Cell Lines ◽  
Annexin V ◽  
Vascular Endothelial ◽  
Caspase 9 ◽  
Migration Assays ◽  
Induced Apoptosis ◽  
Rpmi 8226 ◽  
Anti Tumor Activity

Abstract Abstract 3842 Poster Board III-778 Background and Rationale Vascular disrupting agents (VDAs) act via selectively disrupting established tumor vasculature and have shown remarkable clinical success as anti-cancer therapies. NPI-2358 is a novel VDA with a distinct structure and mechanism of action from other available VDAs. NPI-2358 binds to the colchicine-binding site of beta-tubulin preventing polymerization and disrupting the cytoplasmic microtubule network, thereby causing loss of vascular endothelial cytoskeletal function, and inducing cytotoxicity in cancer cells. Here, we examined the anti-angiogenic and anti-tumor activity of NPI-2358 in multiple myeloma (MM) cells using both in vitro and in vivo model systems. Material and Methods We utilized MM.1S, MM.1R, RPMI-8226, U266, and INA-6 human MM cell lines, as well as purified tumor cells from MM patients relapsing after prior anti-MM therapies. Cell viability/apoptosis assays were performed using MTT, trypan blue exclusion, and Annexin V/PI staining. Angiogenesis was measured in vitro using Matrigel capillary-like tube structure formation assays: Since human vascular endothelial cells (HUVECs) plated onto Matrigel differentiate and form capillary-like tube structures similar to in vivo neovascularization, this assay measures anti-angiogenic effects of drugs/agents. Migration assays were performed using transwell insert assays. Immunoblot analysis was performed using antibodies to caspase-8, caspase-9, caspase-3, PARP, Bcl-2, Bax, pJNK and GAPDH. Statistical significance was determined using a Student t test. Results Treatment of MM.1S, RPMI-8226, MM.1R, INA-6, and KMS-12BM with NPI-2358 for 24h induces a dose-dependent significant (P < 0.005) decrease in viability of all cell lines (IC50 range: 5-8 nM; n=3). To determine whether NPI-2358-induced decrease in viability is due to apoptosis, MM cell lines were treated with NPI-2358 for 24h; harvested, and analyzed for apoptosis using Annexin V/PI staining. A significant increase in NPI-2358-induced apoptosis was observed in all MM cell lines (% Annexin V+/PI- apoptotic cells: MM.1S, 48 ± 2.3%; MM.1R, 46.6 ± 3.1%; RPMI-8226, 61.7 ± 4.5%; and INA-6, 59.9 ± 3.2%; P < 0.05; n=3). Importantly, NPI-2358 decreased viability of freshly isolated MM cells from patients (IC50 range: 3-7 nM; P < 0.005), without affecting the viability of normal peripheral blood mononuclear cells, suggesting specific anti-MM activity and a favorable therapeutic index for NPI-2358. Examination of in vitro angiogenesis using capillary-like tube structure formation assay showed that even low doses of NPI-2358 (7 nM treatment for 12h; IC50: 20 nM at 24h) significantly decreased tubule formation in HUVECs (70-80% decrease; P < 0.05). Transwell insert assays showed a marked reduction in serum-dependent migration of NPI-2358-treated MM cells (42 ± 2.1% inhibition in NPI-2358-treated vs. control; P < 0.05). NPI-2358 at the concentrations tested (5 nM for 12h) in the migration assays did not affect survival of MM cells (> 95% viable cells). A similar anti-migration activity of NPI-2358 was noted against HUVEC cells (48 ± 1.7% decrease in migration; P < 0.05). Mechanistic studies showed that NPI-2358-induced apoptosis was associated with activation of caspase-8, caspase-9, caspase-3 and PARP. Importantly, treatment of MM.1S cells with NPI-2358 (5 nM) triggered phosphorylation of c-Jun amino-terminal kinase (JNK), a classical stress response protein, without affecting Bcl-2 family members Bax and Bcl-2. Blockade of JNK using dominant negative strategy markedly abrogated NPI-2358-induced apoptosis. Conclusion Our preclinical data provide evidence for remarkable anti-angiogenic and anti-tumor activity of NPI-2358 against MM cells, without significant toxicity in normal cells. Ongoing studies are examining in vivo anti-MM activity of NPI-2358 in animal models. Importantly, a Phase-1 study of NPI-2358 as a single agent in patients with advanced malignancies (lung, prostrate and colon cancer) has already established a favorable pharmacokinetic, pharmacodynamic and safety profile; and, a Phase-2 study of the combination of NPI-2358 and docetaxel in non-small cell lung cancer showed encouraging safety, pharmacokinetic and activity data. These findings, coupled with our preclinical studies, provide the framework for the development of NPI-2358-based novel therapies to improve patient outcome in MM. Disclosures: Chauhan: Nereus Pharmaceuticals, Inc: Consultancy. Lloyd:Nereus Pharmaceuticals, In: Employment. Palladino:Nereus Pharmaceuticals, Inc: Employment. Anderson:Nereus Pharmaceuticals, Inc: Consultancy.

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