The Novel Organic Arsenical, Darinaparsin (ZIO-101), Induces Apoptosis through AKT and MEK/ERK Pathways In Hodgkin Lymphoma (HL) and T-Cell Lymphoma (TCL) Cell Lines

Abstract Abstract 2840 Background: The inorganic arsenic, arsenic trioxide (ATO), has a narrow therapeutic index, which has limited its clinical use in most malignancies. Darinaparsin (ZIO-101, S-dimethylarsino-glutathione), synthesized by conjugating dimethylarsenic to glutathione, is a novel organic arsenical that is under investigation as a novel agent for the treatment of cancer. Furthermore, early-phase clinical trials with darinaparsin have demonstrated low toxicity and encouraging clinical efficacy in relapsed/refractory hematologic malignancies. Methods: We treated several TCL cell lines (Jurkat, C10MJ, Hut-78, and MT2) and the resistant HL cell line, L428, with increasing concentrations of darinaparsin (0.5-5μM) +/− the MEK inhibitor, U0126, or ERK siRNA (Qiagen HiPerFect transfection). Cell survival and apoptosis were measured by MTT and Annexin-V/propidium iodide staining, respectively. Further, tumor intracellular darinaparsin and ATO concentrations were assessed with mass spectrometry, while transcription pathway intermediates were analyzed by Western blotting. Results: Darinaparsin inhibited cell growth and induced apoptosis in all cell lines at 1–3μM. At 2μm (48 hours), darinaparsin induced approximately 80% apoptosis in each of the four TCL lines, while 3μM resulted in 65% apoptosis in L428 cells. By comparison, >10μM of ATO (48 hours) was required to induce 40% apoptosis in TCL and 25% apoptosis in L428. At 1–3μM, darinaparsin induced significant increases in caspase 3 and PARP activation in TCL, while interestingly, minimal caspase or PARP was observed in L428. Notably, in L428 cells at 1 hour, mass spectrometry showed that intracellular accumulation of darinaparsin was >10-fold higher as compared with equivalent ATO concentrations (p<0.01). We also treated L428 cells with U0126 (5μM) or ERK2 siRNA, both combined with darinaparsin. Pre-incubation with U0126 or siRNA knock down of ERK2, followed by treatment with darinaparsin, significantly enhanced darinaparsin-induced apoptosis (p<0.05). To further investigate darinaparsin-induced signaling pathways, we analyzed phospho-AKT (p-AKT), and phospho-ERK (p-ERK) in Jurkat and L428. We found down-regulation of p-AKT in Jurkat as well as L428 cells, while total AKT remained unchanged. Additionally, an increase in p-ERK was observed in L428 cells with 2–3μM darinaparsin, while p-ERK was down-regulated in Jurkat cells. Conclusions: Darninaparsin induces significant cell death in HL and TCL cell lines that is mediated through AKT and MEK/ERK-based pathways. Additionally, markedly higher intracellular darinaparsin levels are achieved in lymphoma cells compared with equivalent concentrations of ATO. Continued pre-clinical and clinical trial investigation of darinaparsin in HL and TCL is warranted. Disclosures: No relevant conflicts of interest to declare.

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Motexafin Gadolinium Induces Apoptosis in Lymphoid Cell Lines and Demonstrates Enhanced Biological Activity with Akt Kinase Inhibitors.

Blood ◽

10.1182/blood.v104.11.3406.3406 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3406-3406

Author(s):

Louie Naumovski ◽

Jason Ramos ◽

Jun Chen ◽

Mint Sirisawad ◽

David Lucas ◽

...

Keyword(s):

Cell Death ◽

Chronic Lymphocytic Leukemia ◽

Cell Lines ◽

Specific Inhibitor ◽

Annexin V ◽

Lymphocytic Leukemia ◽

Jurkat Cells ◽

Motexafin Gadolinium ◽

Akt Phosphorylation ◽

Induced Apoptosis

Abstract Motexafin gadolinium (MGd, Xcytrin®) is a tumor-selective redox mediator that catalytically oxidizes intracellular reducing metabolites and produces reactive oxygen species (ROS). In this report, we demonstrate that MGd induces apoptosis or growth inhibition in several hematopoietic tumor-derived cell lines and tumor cells from patients with chronic lymphocytic leukemia. Lymphoma (HF-1, Ramos, DHL-4, DB, Hut78 and Raji) and leukemia (Jurkat, HL-60) cell lines were cultured in RPMI 1640 media with 10% heat inactivated fetal bovine serum with or without 50 uM MGd. MGd inhibited the growth of 6 of the cell lines (HF-1, Ramos, HL-60, DHL-4, Jurkat and DB) and was cytotoxic to HF-1. ROS were implicated in MGd-induced cell death since their presence was detected by dichlorofluorescein diacetate staining and peroxiredoxin oxidation in MGd treated HF-1 cells that undergo apoptosis, but not in Jurkat cells that do not undergo MGd-induced apoptosis. MGd triggered an apoptotic pathway in HF-1 cells as demonstrated by loss of mitochondrial membrane potential, release of cytochrome c from mitochondria, activation of caspases, cleavage of PARP and annexin-V binding. MGd also induced cell death and activated caspases in vitro in primary chronic lymphocytic leukemia cells. Protein lysates from cultured cell lines (HF-1, Ramos) were subjected to immunoblot analysis to determine caspase cleavage patterns, and the phosphorylation status of Akt, a kinase that regulates survival pathways. In MGd treated HF-1, phospho-Akt protein levels initially increased 2–3 fold between 30 min and 1 hr (n=4) and then decreased to 40–50% of control levels by 24–48 hrs (n=4). The drop in phospho-Akt protein coincided with an increase in apoptotic cell death as indicated by morphology, staining with Annexin-V and activation of caspases. Addition of a specific inhibitor of Akt phosphorylation (SH-5) reduced Akt phosphorylation in MGd treated HF-1 cells by 90% and enhanced the cytotoxic effect of MGd. In Ramos cells, which do not undergo apoptosis when treated with MGd, co-treatment with MGd and SH-5 decreased phospho Akt levels by only 15% and did not result in cytotoxicity. These data point to a potential role for Akt in MGd-induced apoptosis and suggest that MGd activity may be enhanced by inhibition of Akt. These data show that the pro-apoptotic effects of MGd involve caspase activation and provide a rationale to evaluate MGd in the treatment of lymphoma and leukemia patients.

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Mir-155 Expression Raises the Apoptotic Threshold in Waldenström Macroglobulinemia By Inhibition of FOXO3a and Bim

Blood ◽

10.1182/blood.v124.21.1671.1671 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1671-1671 ◽

Cited By ~ 1

Author(s):

Brian T Gaudette ◽

Lawrence H. Boise

Keyword(s):

Cell Line ◽

Cell Lines ◽

Conflicts Of Interest ◽

Therapeutic Index ◽

Fold Increase ◽

Malignant Cell ◽

Waldenström Macroglobulinemia ◽

Waldenstrom Macroglobulinemia ◽

Apoptotic Proteins ◽

Induced Apoptosis

Abstract Waldenström Macroglobulinemia (WM) is a proliferative disorder of lymphoplasmacytic cells in the lymph nodes and bone marrow. Phenotypically, WM cells are CD19+, CD20+, CD22+, CD38+, CD138+/- and are lymphoid or plasmacytic in morphology. The disease is characterized by abundant secretion of monoclonal, IgM which causes much of morbidity associated with WM. The disease carries a high prevalence of activating somatic mutations in MyD88 (91%) and CXCR4 (28%), which have been shown to contribute to poor prognosis. These mutations involve signaling cascades that activate pathways known to enhance survival signaling including Bcl-xL. Generally, upregulation of pro-apoptotic Bcl-2 family proteins is observed as cancer cells break differentiation and proliferation checkpoints. To counter this, it becomes necessary for the cell to increase expression of anti-apoptotic Bcl-2 proteins making it dependent on a particular protein or set of proteins for survival. However, we have previously shown data that Bcl-2 family expression in WM is characterized by low expression of both pro- and anti-apoptotic proteins. To investigate a mechanism for this regulation, we examined the Bcl-2 family expression in three WM cell lines and observed that in two lines, BCWM.1 and MWCL-1, the pro-apoptotic BH3-only protein Bim was expressed at very low levels or absent, respectively, which corresponded with low sensitivity to inducers of Bim-dependent intrinsic apoptosis including ABT-737 and dexamethasone. These cell lines were sensitive to bortezomib which can induce apoptosis independent of Bim via a tBid-dependent mechanism. In the third WM cell line, RPCI-WM1, Bim was expressed at moderate levels but the pro-apoptotic proteins Bak and Bax were underexpressed and absent, respectively, which rendered the cell line completely apoptosis-deficient. Having ruled out genomic copy number variation at the loci corresponding to these genes and finding no evidence of epigenetic silencing by methylation, we examined the expression of microRNAs targeting these genes. We first examined the predicted targets of seven commonly dysregulated microRNAs in WM. Of these only one, miR-494, was found to have a moderately conserved target site in the 3’ UTR of Bim. However, the expression pattern of miR-494 did not correlate with the pattern of Bim expression in the WM cell lines. None of these microRNAs were predicted to target Bax or Bak. Therefore, we examined the expression of the remaining commonly dysregulated microRNAs and found that miR-155 was expressed at much higher levels in BCWM.1 and MWCL-1 than in RPCI-WM1 or the multiple myeloma (MM) cell line MM1.s. miR-155 is known to both directly and indirectly regulate FOXO3a, a transcription factor important in the induction of Bim. Confirming this, we observed low protein expression of FOXO3a in both BCWM.1 and MWCL-1 cells. To test this mechanism we stably expressed an anti-miR that targets miR-155 or a control anti-miR in all three WM cell lines and observed an increase in mRNA for FOXO3a and Bim as well as an increase in Bim protein in BWCM.1 and MWCL-1 cells expressing anti-miR-155, while no effect on Bim was observed in the RPCI-WM1 line that does not express miR-155 at high levels. This corresponded with a two-fold increase in ABT-737-induced apoptosis in both BWCM.1 and MWCL-1 in the absence of any additional death signal. As expected, miR-155 antagonism did not significantly increase bortezomib-induced apoptosis. These data indicate that miR-155 expression raises the apoptotic threshold in WM by limiting FOXO3a-mediated Bim expression. Cancer therapy relies on the ability to kill malignant cells at a lower dose than would kill healthy cells. This therapeutic index relies heavily on what is termed mitochondrial priming which is a measure of the expression of pro-apoptotic proteins in a cell. The malignant cell remains alive due to sequestration of these proteins by anti-apoptotic proteins, yet requires less death signaling to cause release of sufficient quantities of pro-apoptotic proteins to activate apoptosis. The data presented here indicate that increased expression of miR-155 raises the apoptotic threshold of WM cells by inhibiting Bim expression and thereby compromises the therapeutic index of many agents. Therefore, the sensitivity to a variety of apoptosis-inducing therapies would be increased by targeting miR-155 in combination as part of the treatment modality. Disclosures No relevant conflicts of interest to declare.

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Birinapant Enhances Bendamustine-Induced Apoptosis In Activated B Cell-Diffuse Large Cell Lymphoma Cells

Blood ◽

10.1182/blood.v122.21.5150.5150 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5150-5150

Author(s):

Indira D Joshi ◽

Mitchell R Smith

Keyword(s):

B Cell ◽

Cell Lines ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

Single Agent ◽

Annexin V ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

Low Level ◽

Induced Apoptosis

Abstract Birinapant (TL32711), a Smac mimetic in clinical testing, potently targets Inhibitor of Apoptosis Proteins (IAPs, including cIAPs and XIAP) to unblock intrinsic and extrinsic pathways, enabling caspase-dependent apoptosis via multiple signals. Birinapant also inactivates canonical NF-kB signaling through cIAPs. We investigated the pro-apoptotic effects of birinapant, alone and in combination with bendamustine (BDM), an active lymphoma therapeutic agent, in a panel of B cell lymphoma cell lines representing germinal center/follicular (GC) vs. activated B cell (ABC) subtypes. We hypothesized that the efficacy of this potential combination therapeutic strategy might differ between GC and ABC lymphoma types, as ABC are reported to be NF-kB-dependent. We used the following EBV negative cell lines: WSU-FSCCL t(14:18)+ follicular lymphoma (FL), FC-TxFL2 t(14:18)+ transformed FL, and SU-DHL4 GC-type diffuse large B cell lymphoma (DLBCL) as examples of GC origin lymphomas. U2932 and TMD8 cell lines represent ABC-type DLBCL. Apoptosis was determined by annexin V staining and confirmed by caspase-3 activation, each assessed by flow cytometric methods following 48 h incubation. Birinapant had little effect (<5% annexin V+ cells) as a single agent on any of these B cell lymphoma cell lines at ≤ 100 nM, though a low level of apoptosis (7-12% annexin V+ cells) was detectable at 10-20 µM in GC types. Addition of birinapant 30-60 minutes prior to BDM did not further enhance the already high level (>50% annexin V+) of apoptosis induced by 10 uM BDM in WSU-FSCCL and FC-TxFL2, and only slightly enhanced the low level of BDM-induced apoptosis in the GC DLBCL cell line DHL-4 (to 10-15%). In the ABC DLBCL cell lines, however, whereas 10uM BDM induced <5% annexin V+ cells for U2932 and 10-15% for TMD8, addition of 100 nM birinapant 30-60 minutes prior to 10 uM BDM induced 35-40% annexin V+ cells in each of these ABC-DLBCL cell lines. This enhancement was schedule-dependent, not observed when birinapant was added after BDM. Thus, the cell lines representing FL and transformed FL are sensitive to BDM at clinically-achievable concentrations, without further enhancement by birinapant. The 3 DLBCL lines were relatively insensitive to BDM compared with FL cells, but BDM-induced apoptosis was markedly enhanced when birinapant was added before (but not after) BDM in the 2 ABC type DLBCL lines. Further explorations into the mechanism of birinapant sensitization of ABC-DLBCL to BDM, issues of dose and schedule, and role of NF-kB-dependency are ongoing. These data suggest that therapeutic trials of BDM plus birinapant would be of interest in ABC type DLBCL. Disclosures: No relevant conflicts of interest to declare.

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Chemosensitivity Is Differentially Regulated by SDF-1/CXCR4 and SDF-1/CXCR7 Axes in Acute Lymphoblastic Leukemia.

Blood ◽

10.1182/blood.v120.21.2441.2441 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2441-2441

Author(s):

Norie Ando ◽

Yoshiyuki Furuichi ◽

Shin Kasai ◽

Keiko Kagami ◽

Kumiko Goi ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Cell Lines ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Annexin V ◽

Cxcr4 Antagonist ◽

Hematopoietic Stem ◽

Lower Chamber ◽

Induced Apoptosis ◽

Cxcr4 Axis

Abstract Abstract 2441 SDF-1 (CXCL12) is a chemokine vastly secreted from bone marrow-stromal cells (BM-SC), and plays a pivotal role for multiple biological events in a variety of cells. Hematopoietic stem cells express its receptor CXCR4 at high levels and the SDF-1/CXCR4 system is profoundly implicated in their homing to BM-SC resulting in their chemoresistance. Although important roles of the SDF-1/CXCR4 system for acute myeloid leukemia (AML) have been extensively characterized, its biological implication in acute lymphoblastic leukemia (ALL) remains not fully elucidated. Recently, an alternative SDF-1 receptor CXCR7 was identified, but its functional role in ALL is almost unclear. In the present study, we compared the SDF-1/CXCR4 and SDF-1/CXCR7 systems, particularly about effects on induction of anti-leukemic agent-induced apoptosis, by using established ALL cell lines with MLL gene rearrangement (n=9) and inhibitors against CXCR4 and CXCR7 respectively. Surface CXCR4 expression was very high in all of the cell lines, while surface CXCR7 expression was limited regardless of its abundant cytoplasmic expression. Neither addition of CXCR4 antagonist AMD3100 (1μM) nor CXCR7 antagonist CCX733 (250nM) in culture media showed a significant effect on proliferation of cell lines, suggesting that both systems have not an essential role in proliferation. The chemotactic assay using the Transwell showed a marked migration of cells from the upper to the lower chamber when SDF-1 (10–100nM) or confluent BM-SC line KM104 was present in the lower chamber, and its elicited migration was almost canceled by pretreatment with AMD3100, but only very modestly by pretreatment with CCX733. Next, changes in sensitivity to cytosine arabinoside (Ara-C) after pretreatment of cell lines with either of antagonists was examined on flow cytometry after 48-hour incubation of Ara-C (100nM). Of importance, the Annexin V-labeled apoptotic population with AMD3100 (1μM) pretreatment was significantly decreased as compared with that without pretreatment. It should be noted that when myeloid HL60 cells were used in this experiment, AMD3100 pretreatment rather enhanced Ara-C-induced apoptosis. To address molecular mechanism of AMD3100-induced chemoresistance, the chip analysis was performed and revealed that 24-hour AMD3100 plus Ara-C treatment induced a decrease in RNAs of several caspases and pro-apoptotic molecules as compared with Ara-C alone. In contrast, the Annexin V-labeled apoptotic population with CCX733 pretreatment was significantly increased in a dose-dependant manner of CCX733 (10–500nM) as compared with that without pretreatment. Taken together, SDF-1/CXCR4 and SDF-1/CXCR7 axes are differentially implicated in chemosensitivity of ALL cells that are not adherent to BM-SC; up-regulation of sensitivity via SDF-1/CXCR4 axis vs. down-regulation of sensitivity via SDF-1/CXCR7 axis. Of clinical importance, in contrast to AML, blockade of the SDF-1/CXCR4 axis in ALL facilitate chemoresistance, suggesting that a clinical application of CXCR4 antagonist currently undergoing in AML patients should be carefully considered in ALL patients. Disclosures: No relevant conflicts of interest to declare.

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Inhibition of the Myocardin-Related Transcription Factor Pathway Increases Efficacy of Trametinib in NRAS-Mutant Melanoma Cell Lines

Cancers ◽

10.3390/cancers13092012 ◽

2021 ◽

Vol 13 (9) ◽

pp. 2012

Author(s):

Kathryn M. Appleton ◽

Charuta C. Palsuledesai ◽

Sean A. Misek ◽

Maja Blake ◽

Joseph Zagorski ◽

...

Keyword(s):

Cell Lines ◽

Melanoma Cell ◽

Therapeutic Potential ◽

Mapk Pathway ◽

Mek Inhibitor ◽

Intrinsic Resistance ◽

Primary Resistance ◽

Induced Apoptosis ◽

Melanoma Cell Lines

The Ras/MEK/ERK pathway has been the primary focus of targeted therapies in melanoma; it is aberrantly activated in almost 80% of human cutaneous melanomas (≈50% BRAFV600 mutations and ≈30% NRAS mutations). While drugs targeting the MAPK pathway have yielded success in BRAFV600 mutant melanoma patients, such therapies have been ineffective in patients with NRAS mutant melanomas in part due to their cytostatic effects and primary resistance. Here, we demonstrate that increased Rho/MRTF-pathway activation correlates with high intrinsic resistance to the MEK inhibitor, trametinib, in a panel of NRAS mutant melanoma cell lines. A combination of trametinib with the Rho/MRTF-pathway inhibitor, CCG-222740, synergistically reduced cell viability in NRAS mutant melanoma cell lines in vitro. Furthermore, the combination of CCG-222740 with trametinib induced apoptosis and reduced clonogenicity in SK-Mel-147 cells, which are highly resistant to trametinib. These findings suggest a role of the Rho/MRTF-pathway in intrinsic trametinib resistance in a subset of NRAS mutant melanoma cell lines and highlight the therapeutic potential of concurrently targeting the Rho/MRTF-pathway and MEK in NRAS mutant melanomas.

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

BioMed Research International ◽

10.1155/2019/7298539 ◽

2019 ◽

Vol 2019 ◽

pp. 1-18 ◽

Cited By ~ 3

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.

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Deguelin Induces Apoptosis by Targeting Both EGFR-Akt and IGF1R-Akt Pathways in Head and Neck Squamous Cell Cancer Cell Lines

BioMed Research International ◽

10.1155/2015/657179 ◽

2015 ◽

Vol 2015 ◽

pp. 1-9 ◽

Cited By ~ 17

Author(s):

Yuh Baba ◽

Masato Fujii ◽

Toyonobu Maeda ◽

Atsuko Suzuki ◽

Satoshi Yuzawa ◽

...

Keyword(s):

Growth Factor ◽

Head And Neck ◽

Cell Lines ◽

Squamous Cell ◽

Cell Cancer ◽

Growth Factor Receptor ◽

Annexin V ◽

Akt Activation ◽

Igf1r Signaling ◽

Induced Apoptosis

Deguelin, a rotenoid compound from the African plantMundulea sericea(Leguminosae), has been shown to possess antitumor activities but the exact role for the growth factor receptor mediated signaling pathway in head and neck squamous cell carcinoma (HNSCC) is currently still unclear. In the present study, we investigated the effect of deguelin on epidermal growth factor receptor (EGFR) and insulin-like growth factor-1 receptor (IGF1R) pathways in HNSCC cell lines. Flowcytometric analysis revealed accumulation of annexin V positivity in deguelin-treated cells, showing that deguelin induced apoptosis. The deguelin-induced apoptosis was accompanied by the reduction of constitutive phosphorylated levels of IGF1R, Akt, and extracellular signal-regulated kinase1/2 (ERK1/2). LY294002-mediated inhibition of phosphatidylinositol-3 kinase, which is an upstream effector for Akt activation, increased cleavage of poly(ADP-ribosyl) polymerase (PARP) but ERK inhibition by U0126 did not. Deguelin inhibited both IGF-1- and EGF-induced Akt activation. These results showed that deguelin possessed antitumor effect by targeting Akt in dual axis such as EGFR and IGF1R signaling pathways and suggested that it provides an applicable therapeutic strategy for HNSCC patients.

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

Blood ◽

10.1182/blood.v108.11.3480.3480 ◽

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.

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

Blood ◽

10.1182/blood.v114.22.3842.3842 ◽

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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Induction of Apoptosis In Imatinib-Resistant BCR-ABL1-Positive Leukemia Cell Lines by Bortezomib

Blood ◽

10.1182/blood.v116.21.4469.4469 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4469-4469

Author(s):

Hilmar Quentmeier ◽

Sonja Eberth ◽

Julia Romani ◽

Margarete Zaborski ◽

Hans G. Drexler

Keyword(s):

Cell Lines ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Philadelphia Chromosome ◽

Leukemia Cell ◽

Resistant Cell ◽

Imatinib Resistance ◽

Imatinib Resistant ◽

Induced Apoptosis

Abstract Abstract 4469 The BCR-ABL1 translocation occurs in chronic myeloid leukemia (CML) and in 25% of cases with acute lymphoblastic leukemia (ALL). We screened a panel of BCR-ABL1 positive cell lines to find models for imatinib-resistance studies. Five of 19 BCR-ABL1 positive cell lines were resistant to imatinib-induced apoptosis (KCL-22, MHH-TALL1, NALM-1, SD-1, SUP-B15). None of the five resistant cell lines carried mutations in the kinase domain of BCR-ABL1 and – consequently – all also showed resistance to the second generation kinase inhibitors, nilotinib or dasatinib. All Philadelphia chromosome (Ph)-positive cell lines demonstrated constitutive phosphorylation of STAT5 and S6. Imatinib induced dephosphorylation of both BCR-ABL1 downstream effectors in responsive cell lines, but - remarkably – induced dephosphorylation of STAT5 in resistant cell lines as well. By administering well-described signalling pathway inhibitors we were able to show that activation of mTOR complex 1 was responsible for the constitutive S6 phosphorylation of imatinib-resistant cells. Neither BCR-ABL1 nor Src kinases or Ras/Rac-GTPases underlie tyrosine kinase inhibitor resistance in these cell lines. In conclusion, none of the five TKI-resistant cell lines showed aberrant activation of previously-described oncogenic pathways which would explain their resistance. These findings raise the question whether these cell lines might help to find a novel – alternative – explanation for TKI resistance. Interestingly, the proteasome inhibitor bortezomib induced apoptosis in TKI-resistant and –sensitive Ph+ cell lines. Bortezomib is being used for the treatment of multiple myeloma. Our findings support the notion that bortezomib might also be useful for the treatment of imatinib-resistant CML. Disclosures: No relevant conflicts of interest to declare.

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