The Tricyclic Coumarin GUT-70 Induces Apoptosis and Cell Cycle Arrest Preferentially in Mantle Cell Lymphomas with Mutant p53.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1684-1684
Author(s):  
Linhua Jin ◽  
Shinya Kimura ◽  
Yixin Zhou ◽  
Junya Kuroda ◽  
Hiroya Asou ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Anticancer Agent ◽  
Cell Lymphoma ◽  
Annexin V ◽  
B Cell Lymphoma ◽  
S Phase ◽  
26S Proteasome ◽  
Mutant P53 ◽  
Mantle Cell

Abstract Abstract 1684 Poster Board I-710 Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma resistant to standard chemotherapy. Since p53 inactivating mutations occur primarily in the aggressive and refractory MCL variants, development of novel compounds that target p53-independent signaling pathways is of considerable interest. We investigated the cytotoxic efficacy and molecular mechanisms of a newly discovered anticancer agent GUT-70 (synthesized at Nippon Shinyaku, Kyoto, Japan), a natural product derived from the stem bark of Calophyllum brasiliense, characterized as a tricyclic coumarin with the formula 5-methoxy-2,2-dimethyl-6-(2-methyl-1-oxo-2-butenyl) -10-propyl-2H,8H-benzo[1,2-b;3,4-b]dipyran-8-one (C23H26O5). This agent has pronounced anti-tumor activity, but does not inhibit colony formation by normal hematopoietic progenitors or proliferation of normal human hepatocytes. (Kimura, Int J Cancer 2005;113:158) However, their mechanisms have not been fully investigated. In this study, cytotoxicity and mechanisms of action of GUT-70 were investigated in MCL cell lines with wild-type and mutant p53 (wt-p53: JVM-2, Granta-519, mt-p53: Jeko-1, MINO). Treatment with GUT-70 resulted in marked reduction in cell growth (trypan blue corrected cell numbers) and an increase in the apoptotic fraction (Annexin V), in a time- and concentration-dependent manner. Importantly, mt-p53 MCL were more sensitive than wt-p53 cells (IC50 at 48 hrs: JVM-2, 4.5 μM; Granta 519, 6.3 μM; Jeko-1, 0.7 μM; MINO, 2.2 μM, % specific apoptosis of 5μM GUT-70 treated cell: JVM-2, 18.5%; Granta 519, 17.6%; Jeko-1, 38.1%; MINO, 30.9%; Annexin V). GUT-70 also impeded cell cycle progression, resulting in a decreased S-phase with increased G0/G1 cells independent of p53 status (S-phase was decreased by 8.2 % in JVM-2, 12.1% in Granta 519, 10.0 % in Jeko-1, 9.8 % in MINO). This was associated with a dramatic morphological change: bleb-like cytoplasmic enlargement without visible nuclear breakdown observed by phase-contrast time-lapse video microscopy. Next, the ability of GUT-70 to modulate cell cycle and apoptosis related proteins including p53 target genes was analyzed by western blotting. GUT-70 treatment significantly reduced cyclin D1, the hallmark of MCL, believed to be critical for lymphomagenesis, and increased p27 levels. Furthermore, GUT-70 inactivated and/or degraded Rb and repressed E2F1, effects similar to the action of the specific 26S proteasome inhibitors MG132 and bortezomib. GUT-70 induced mitochondrial apoptosis associated with caspase-9 and -3 activation, accompanied by transcriptional induction of the proapoptotic BH3-only protein Noxa. Notably, in highly sensitive Jeko-1 and MINO cells expressing mt-TP53, antiapoptotic Mcl-1 was not upregulated, whereas in less sensitive JVM-2 and Granta-519 cells with wt-TP53 GUT-70 caused Mcl-1 accumulation, which co-immunoprecipitated with Noxa. In addition, we observed higher levels of activated Bak in Jeko-1 and MINO cells compared to JVM-2 and Granta-519 cells. In summary, these data indicate that the novel anticancer agent GUT-70 depletes cyclin D1 and induces mitochondrial apoptotic cell death in MCL. Notably, these effects are more pronounced in MCL with mutant p53, a known negative prognostic factor for MCL. These findings suggest potential utility of GUT-70 for the treatment of MCL. Disclosures No relevant conflicts of interest to declare.

Targeting the Cyclin D - CDK4/6 - Rb Axis in Mantle Cell Lymphoma with the Novel Translation Inhibitor Silvestrol,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3498-3498
Author(s):  
Lapo Alinari ◽  
Ryan B. Edwards ◽  
Courtney J. Prince ◽  
William H. Towns ◽  
Rajeswaran Mani ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Tumor Cells ◽  
Cell Lymphoma ◽  
S Phase ◽  
Cyclin D ◽  
Mantle Cell ◽  
Phase Entry

Abstract Abstract 3498 During cell cycle progression, D class cyclins activate cyclin dependent kinases (CDK) 4 and 6 to phosphorylate and inactivate Rb, allowing E2F-1 mediated transcription of additional cell cycle genes including cyclin E to drive S phase entry. This critical pathway is nearly universally dysregulated in cancer, providing tumor cells a strong growth advantage and escape from normal mitotic control. Substantial research is being directed toward targeting this pathway in many cancer types, with some preliminary successes being achieved with pharmacologic inhibitors of CDK4/6. However the development of alternative strategies to block this pathway could potentially provide broad therapeutic benefit. A prime example of a tumor with a disrupted cyclin D axis is Mantle Cell Lymphoma (MCL), in which the t(11;14) translocation places CCND1, the gene for cyclin D1, under the control of an immunoglobulin promoter. This results in sustained cyclin D1 expression in tumor cells and concomitant Rb inactivation, S phase entry and cell division. MCL is a relatively uncommon subset of Non-Hodgkin Lymphoma, but accounts for a disproportionate number of deaths. Treatments are limited and relapse is nearly universal; thus, new treatment strategies are essential for this disease. Silvestrol is a structurally unique, plant-derived cyclopenta[b]benzofuran with potent in vitro and in vivo anti-tumor activity in several model systems including B-cell acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL). Silvestrol inhibits the initiation step of translation by preventing assembly of eIF4A and capped mRNA into the eIF4F complex, leading to selective loss of short half-life proteins such as Mcl-1 and cyclin D1. We therefore hypothesized that silvestrol, through the depletion of cyclin D1, would demonstrate efficacy in MCL. Silvestrol showed low nanomolar IC50 values in the JeKo-1 (13 nM), Mino (17 nM) and SP-53 (43 nM) MCL cell lines at 48 hr (MTS assay; cell death confirmed by propidium iodide flow cytometry). This potency was similar in primary MCL tumor cells. Longer exposure times substantially improved the cytotoxicity of silvestrol assessed at 48 hr (approximately 50% effect achieved with a 16 hr exposure vs. 80% effect with a 24 hr exposure), suggesting that the cellular impacts of this agent increase with exposure time. Cyclins D1 and D3 were dramatically reduced in MCL cell lines with just 10 nM silvestrol at 16 hr (cyclin D2 was undetectable in these cells), with subsequent loss of Rb phosphorylation as well as cyclin E mRNA and protein, culminating in G1 cell cycle arrest. Similar to what we previously showed in CLL and ALL cells, silvestrol treatment under these conditions also caused loss of Mcl-1 protein with concurrent mitochondrial depolarization, although the exact mechanism of silvestrol-mediated cytotoxicity in these cells is still under investigation. In an aggressive xenograft mouse model of MCL, silvestrol produced a highly significant improvement in survival [median survival of vehicle vs. silvestrol treated mice (1.5 mg/kg every 48 hr) = 27 vs. 38 days; P<0.0001] without detectable toxicity. Together, these data demonstrate that the translation inhibitor silvestrol has promising in vitro and in vivo activity in MCL preclinical models. Furthermore, as the cyclin D/CDK/Rb axis is disrupted in most tumor types, this strategy may be broadly effective in other cancers as well. Disclosures: No relevant conflicts of interest to declare.

SOX11 is useful in differentiating cyclin D1-positive diffuse large B-cell lymphoma from mantle cell lymphoma

2012 ◽  
Vol 61 (4) ◽  
pp. 685-693 ◽  
Author(s):  
Shih-Chuan Hsiao ◽  
Inmaculada Ribera Cortada ◽  
Luis Colomo ◽  
Hongtao Ye ◽  
Hongxiang Liu ◽  
...  
Keyword(s):  
B Cell ◽  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Large B Cell Lymphoma ◽  
Mantle Cell ◽  
Large B Cell

Development of B Cell Lymphoma in Eμ-Cyclin D1 X Mutant p53 Transgenic Mice.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1907-1907
Author(s):  
Mitchell R. Smith ◽  
Indira J. Joshi ◽  
Fang Jin ◽  
Tahseen Al-Saleem
Keyword(s):  
T Cell ◽  
Transgenic Mice ◽  
B Cell ◽  
Cyclin D1 ◽  
Cell Line ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Mutant P53 ◽  
B Cell Lymphomas ◽  
T Cell Lymphomas

Abstract Background: Mantle cell lymphoma (MCL) is characterized by t(11;14) which dysregulates cyclin D1 expression. Eμ-cyclinD1 transgenic mice, however, are healthy. Additional genetic events must be necessary for lymphomagenesis, and knowledge of these would enhance understanding and therapy of MCL. In addition, a mouse model of MCL would be helpful in drug development. Alterations in p53 have been described in MCL, often associated with the blastic variant. Objectives and Methods: To determine whether p53 and cyclin D1 can cooperate in lymphomagenesis, we cross bred Eμ-cyclinD1 transgenic mice (Bodrug et al EMBO J, 1996, courtesy of Alan Harris) with mice transgenic for mutant p53 (Jackson Labs, Jacks et al Curr Biol, 1994). Progeny mice were monitored for presence of the transgenes by PCR of tail vein DNA and observed for development of disease. Results: Of mice carrying both aberrant genes, 24 of 38 developed B cell lymphoma. Mice did not become visibly ill until at least 12 months of age, with median age at sacrifice 15.5 (range 12–23) months. The lymphoma was generally disseminated, involving spleen, liver, diffuse adenopathy and marrow with occasional extranodal sites. Histology varied between small and large cell, with some having a vaguely follicular growth pattern. T cell lymphomas occurred in 2 other mice, while 5 developed osteosarcoma (1 of these in a mouse that also had B cell lymphoma). The B cell lymphomas were clonal by Cμ-VH PCR. Cyclin D1 expression was documented by Western analysis. A cell line has also been developed from one of the B cell lymphomas and this line rapidly grows into disseminated lymphoma in syngeneic mice. These B cell lymphomas differ from the thymic T cell lymphomas seen in heterozygous p53 mutant mice that do not co-express cyclin D1. The latency period differs from cyclin D1 x myc double transgenic mice. Conclusions: This model demonstrates cooperation between p53 and cyclin D1 pathways in B cell lymphomagenesis and should prove useful in delineating how these signals interact. The cell line may prove useful in pre-clinical testing of new agents for MCL.

Iron Chelation in Mantle Cell Lymphoma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1395-1395
Author(s):  
Heather M. Gilbert ◽  
Josef T. Prchal ◽  
Miles C. Deneris
Keyword(s):  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Cell Lymphoma ◽  
Iron Chelation ◽  
B Cell Lymphoma ◽  
Iron Chelators ◽  
Large B Cell Lymphoma ◽  
Mantle Cell ◽  
Lymphoma Line ◽  
Large B Cell

Abstract Cell proliferation is dependent upon iron, and numerous studies have shown that iron limitation arrests cells in the G1 phase of the cell cycle. A recent study of the molecular basis of these observations (Richardson, et al. Blood2007;109:4045) examined the ability of iron chelators to inhibit cell proliferation and to induce apoptosis, focusing on the role of iron chelation on cyclin D1. Cyclin D1 assembles with cdk-4 or cdk-6, generating an active holo-enzyme that catalyzes a rate limiting step in G1/S progression. This complex phosphorylates substrates, including the retinoblastoma protein, which regulate S phase entrance. Richardson’s group demonstrated that the G1/S arrest after Fe depletion is mediated, in part, by a decrease in cyclin D1 via ubiquitin-independent proteasomal degradation. Studies looking specifically at mantle cell lymphoma cell lines, however, have not yet been reported. Mantle Cell lymphoma is an interesting target for potential iron chelation as it is associated with a balanced translocation (t11;14) which leads to upregulation of BCL1 and to the constitutive overproduction of cyclin D1. We studied five different cell lines - JeKo (Mantle Cell Lymphoma), BL-41 (Burkitt Cell Lymphoma), DG-75 (Burkitt Cell Lymphoma), SUDHL-6 (Diffuse Large B cell Lymphoma) and EBV-immortalized lymphocytes from normal controls - and incubated them with four different iron chelators - deferoxamine (DFO), 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311), Pyridoxal Isonicotinoyl Hydrazone (PIH), and Salicylaldehyde Isocotinoyl Hydrazone (SIH). We then measured and compared cell cycle proliferation (using the Cellometer Auto T4, an instrument that measures cell count, cell viability, and cell size) and the rate of apoptosis (via propidium iodide FACS analysis). At 24 hours incubation, the mantle cell lymphoma lines showed significantly increased rates of apoptosis compared with non-chelated mantle cell controls (5% vs. 48%, p=0.04). The diffuse large B cell lymphoma line showed a lesser increase in apoptosis that did not reach statistical significant (6.5% vs. 14%, p=0.07), while the Burkitt’s lymphoma lines and the EBV immortalized lymphocytes showed no significant difference (BL-41, 3.4% vs. 4.1%, p=0.50; DG-75, 6% vs. 5.9%, p=0.99; EBV lymphocytes, 12.5% vs. 12.7%, p=0.96). At 72 hours of incubation with chelators, the EBV lymphocytes showed increased apoptosis compared to untreated controls (2.5% vs. 44.5%, p=0.002), while the apoptotic rate increased in the diffuse large B cell lymphoma line (3.8% vs. 48%, p=0.001) and even more dramatically in the mantle cell lymphoma line (1.5% vs. 64%, p=0.0006). The two Burkitt’s lymphoma lines were affected to a lesser degree at 72 hours by the presence of iron chelators (BL-41, 0.9% vs, 3.9%, p=0.02; DG-75, 5.5% vs. 8.9%, p=0.11). Although iron chelation, especially at longer incubation times, did affect all cell lines to various degrees, the chelator-mediated effects do appear to be specific for cell type, with mantle cell lymphoma cells displaying higher rates of apoptosis compared with other lymphomas and normal lymphocytes. These initial results will now be followed by examination of cyclin D1 expression after iron chelation. If overexpression of cyclin D1 in mantle cell lymphoma releases cells from their normal controls and acts as an oncogene, then a decrease in cyclin D1 levels via iron chelation could be added to the therapeutic armamentarium of mantle cell lymphoma.

Cell Cycle Regulation by Iron: Novel Approaches to Mantle Cell Lymphoma Therapy.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2515-2515 ◽  
Author(s):  
Heather Gilbert ◽  
John Cumming ◽  
Josef T. Prchal
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Iron Chelation ◽  
Lymphoma Cell ◽  
Protein Levels ◽  
Mantle Cell

Abstract Abstract 2515 Poster Board II-492 Mantle cell lymphoma is a well defined subtype of B-cell non-Hodgkin lymphoma characterized by a translocation that juxtaposes the BCL1 gene on chromosome 11q13 (which encodes cyclin D1) next to the immunoglobulin heavy chain gene promoter on chromosome 14q32. The result is constitutive overexpression of cyclin D1 (CD1) resulting in deregulation of the cell cycle and activation of cell survival mechanisms. There are no “standard” treatments for MCL. Despite response rates to many chemotherapy regimens of 50% to 70%, the disease typically progresses after treatment, with a median survival time of approximately 3-4 years. Mantle cell lymphoma represents a small portion of malignant lymphomas, but it accounts for a disproportionately large percentage of lymphoma-related mortality. Novel therapeutic approaches are needed. In 2007, Nurtjaha-Tjendraputra described how iron chelation causes post-translational degradation of cyclin D1 via von Hippel Lindau protein-independent ubiquitinization and subsequent proteasomal degradation (1). Nurtjaha-Tjendraputra demonstrated that iron chelation inhibits cell cycle progression and induces apoptosis via proteosomal degradation of cyclin D1 in various cell lines, including breast cancer, renal carcinoma, neuroepithelioma and melanoma. Our preliminary data show similar findings in mantle cell lymphoma. To establish whether iron chelation can selectively inhibit and promote apoptosis in mantle cell derived cell lines, the human MCL cell lines Jeko-1, Mino, Granta and Hb-12; the Diffuse Large B cell lymphoma line SUDHL-6; and the Burkitt's Lymphoma lines BL-41 and DG75 were grown with media only, with two different iron chelators (deferoxamine (DFO) and deferasirox) at various concentrations (10, 20, 40, 100 and 250 μM), and with DMSO as an appropriate vehicle control. Cells were harvested at 24, 48 and 72 hours. For detection of apoptotic cells, cell-surface staining was performed with FITC-labeled anti–Annexin V antibody and PI (BD Pharmingen, San Diego, CA). Cell growth was analyzed using the Promega MTS cytotoxicity assay. CD1 protein levels were assessed using standard Western blot techniques. At 24, 48 and 72 hours of incubation with iron chelators, the mantle cell lymphoma cell lines showed significantly increased rates of apoptosis compared to the non-mantle cell lymphoma cell lines (p<0.0001 for all time points). DFO and deferasirox inhibted cell growth with an IC50 of 18 and 12 μM respectively. All of the mantle cell lines had measurable cyclin D1 levels at baseline. None of the non-mantle cell lines expressed baseline measurable cyclin D1. In the mantle cell lines, cyclin D1 protein levels were no longer apparent on western blot after 24 hours of incubation with chelation. We then added ferrous ammonium sulfate (FAS) to DFO in a 1:1 molarity ratio and to deferasirox in a 2:1 ratio, and then treated the same lymphoma cell lines with the FAS/chelator mixture and with FAS alone for 72 hours. Adding iron to the chelators completely negated all the pro-apoptotic effects that were seen with iron chelation treatment. Treating with FAS alone had no effect on cell growth or apoptosis. Iron chelation therapy with both DFO and deferasirox results in decreased cell growth, increased cellular apoptosis, and decreased cyclin D1 protein levels in vitro in mantle cell lymphoma. The cytotoxic effects are prevented by coincubation with ferrous ammonium citrate, confirming that the effects are due to iron depletion. Proposed future research includes further defining the molecular basis of iron chelation effects; studying these therapies in combination with other cancer treatments both in vitro and in vivo; and studying iron chelation therapy in mantle cell lymphoma patients. 1. Nurtjahja-Tjendraputra, E., D. Fu, et al. (2007). “Iron chelation regulates cyclin D1 expression via the proteasome: a link to iron deficiency-mediated growth suppression.” Blood109(9): 4045–54. Disclosures: No relevant conflicts of interest to declare.

FTY720 Demonstrates Promising in-Vitro and in-Vivo Pre-Clinical Activity by Down-Modulation of Cyclin D1 and Pakt in Mantle Cell Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3728-3728
Author(s):  
Lapo Alinari ◽  
Qing Liu ◽  
Ching-Shih Chen ◽  
Fengting Yan ◽  
James T Dalton ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Cell Line ◽  
Cell Lymphoma ◽  
Tumor Burden ◽  
Time Dependent ◽  
Control Group ◽  
Mantle Cell

Abstract Abstract 3728 Poster Board III-664 Over-expression of Cyclin D1 and constitutive phosphorylation of Akt has been implicated in the pathogenesis of mantle cell lymphoma (MCL). Here we describe FTY720 (fingolimod), an immunosuppressive agent currently being explored in phase III studies in renal transplantation and multiple sclerosis patients, to mediate time- and dose-dependent cell death in primary MCL cells (6 patients) and MCL cell lines, Jeko and Mino. FTY720-induced apoptosis was associated with reactive oxygen species (ROS) generation, Bax up-regulation but not associated with caspase 3 activation in MCL. FTY720 treatment resulted in time-dependent down-modulation of Cyclin D1 and phospho Akt (p-Akt) protein level, two critical disease-relevant molecules in the pathogenesis of MCL. Consistent with the modulation of Cyclin D1, FTY720-induced cell cycle arrest with accumulation of cells in G0/G1 and G2/M phases of the cell cycle with concomitant decrease in S phase entry. Importantly, FTY720 treatment was also associated with a time-dependent phospho Erk (p-Erk) induction in Mino and Jeko cells. To determine the in vivo efficacy of FTY720, we developed a preclinical, in vivo xenograft model of human MCL where MCL cell lines (Jeko, Mino and SP53) were engrafted into severe combined immune deficient (SCID) mice. Cell dose titration trials identified 4 × 107 Mino or Jeko cells injected intravenously via tail vein to result in consistent engraftment and fatal tumor burden in all mice. All mice engrafted with 4 × 107 Jeko cells developed a disseminated disease within 3 weeks and had a median survival of 28 days (compared to 43 days for Mino and 51 days for SP53). Because the Jeko cell line was established from the peripheral blood of a patient with blastic variant MCL and demonstrated a more resistant phenotype to several immuno-chemoterapeutic compounds, this cell line was chosen to create a more stringent in vivo preclinical model. SCID mice were treated with the monoclonal antibody TMβ1 to deplete murine NK cells, engrafted with 4 × 107 Jeko cells and observed daily for signs of tumor burden. Ten mice/group were treated starting at day 15 post-engraftment with intraperitoneal injection of 100 μl of saline or FTY720 (5 mg/kg resuspended in 100 μl of saline), every day, for two weeks. The median survival for FTY720-treated mice (N=10) was 38 days (95% CI:30-39) compared to 26.5 days (95% CI: 26-27 days) for the control group mice (N=10). The results from the log-rank test indicated an overall statistical significant difference in survival functions between the FTY720 treatment and the control group (p=0.001). These results provide the first evidence for a potential use of FTY720 in targeting key pathways that are operable in the pathogenesis of MCL and warrant the further investigation of FTY720 in combination with other agents in clinical trials treating patients with MCL. Disclosures: No relevant conflicts of interest to declare.

The Bioactive Polyphenol Curcumin (diferuloylmethane) In Human Apolipoprotein A-1 Nanodisks Enhances Apoptosis and G1 Cell Cycle Arrest In Mantle Cell Lymphoma Compared with Free Curcumin

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3934-3934
Author(s):  
Amareshwar T.K. Singh ◽  
Mistuni Ghosh ◽  
C. Shad Thaxton ◽  
Trudy M. Forte ◽  
Robert O. Ryan ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Drug Delivery ◽  
Cyclin D1 ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Parp Cleavage ◽  
Cycle Arrest ◽  
Mantle Cell ◽  
Induced Apoptosis

Abstract Abstract 3934 Background: Mantle cell lymphoma (MCL) is a pre–germinal center neoplasm characterized by cyclin D1 overexpression resulting from translocation of the cyclin D1 gene on 11q13 to the promoter of the immunoglobulin heavy chain locus on 14q32. Since MCL is incurable with standard lymphoma therapies, new treatment approaches are needed that target specific biologic pathways. The bioactive polyphenol curcumin (Curc), derived from the rhizome of Curcuma longa Linn, has been shown to have pleiotropic activities related to its complex chemistry and its influence on multiple signaling pathways including NF-kB, Akt, Nrf2 and pathways involved in metastasis and angiogenesis. Curc has been shown to cause growth arrest and apoptosis of BKS-2 immature B-cell lymphoma by downregulating growth and survival promoting genes (Clin Immunol 1999; 93:152). However, because of poor aqueous solubility Curc has had limited clinical utility, so investigators have explored nanoparticle drug delivery approaches (J Nanobiotech 2007, 5:3, MCT 2010; 9:2255). We reasoned that effective and targeted drug delivery by nanoparticles required appropriate receptors to facilitate binding. We therefore screened lymphoma cell lines for receptors that recognize apolipoprotein (apo) A-1. We hypothesized that a novel discoidal nanoparticle (ND) consisting of apoA-1, phospholipid and Curc (Curc ND) would bind to such receptors to facilitate drug delivery. Methods: We compared biologic activity of free Curc vs. Curc-ND in MCL cell lines expressing receptors for apoA-1. Cell lines were grown and maintained in culture, treated, and apoptosis and cell cycle progression was measured by flow cytometry. Relevant signaling intermediates and presence of apoA-1 receptors were measured by immunoblotting using specific antibodies. Results: Granta and Jeko cells (both MCL cell lines) expressed apoA-1 receptors including class B scavenger receptor (SR-B1) and the ATP-binding cassette transporter of the sub-family G1 (ABCG1). To compare the pro-apoptotic effect of free Curc and Curc-ND, Granta cells were incubated with free Curc, Curc-ND, empty ND, and medium alone (untreated). Compared to medium alone, empty ND had no effect while free Curc (20 μM) induced apoptosis. Curc-ND produced a dose-dependent increase in apoptosis, with ∼70% apoptosis at 20 μM. To investigate the mechanism of Curc-ND induced apoptosis, apoptosis-related proteins were studied in cultured Granta cells. A time-dependent decrease in caspase-9 levels was observed following incubation with Curc-ND or free Curc. The decrease in caspase-9 seen with Curc-ND, however, occurs much earlier (between 2–4 h of incubation) than for free-Curc. Caspase-3 was undetectable after 16 h with either treatment. Loss of this band implies activation of caspase-3, which was confirmed by PARP cleavage, wherein a decrease in the 116 kD band was accompanied by an increase in the 85 kD cleavage product. Unlike free Curc, Curc-ND induced PARP cleavage even at 16 h of incubation, suggesting sustained drug release. Curc-ND downregulated cyclin D1, decreased Akt phosphorylation and enhanced cleavage of caspases-9 and -3, and PARP. In addition, Curc-ND induced G1 cell cycle arrest to a greater extent than free Curc in Granta and Jeko cells (Granta: Control 34% G1, Curc 37% G1, Curc-ND 46% G1; Jeko: Control 39% G1, Curc 49% G1, Curc-ND 54% G1). Conclusion: We have shown that the MCL cell lines Granta and Jeko express apoA-1 receptors, making them likely targets for discoidal nanoscale delivery vehicles stabilized with Apo-A1. These nanodisks, when carrying the polyphenol Curc, can result in increased caspase -dependent apoptosis, cell cycle arrest, downregulation of cyclin-D1 and decreased p-Akt. These data suggest that the pleiotropic polyphenol Curc has cell killing/arrest activity in MCL and that Curc-ND may be a potential therapeutic with drug targeting ability. Disclosures: Forte: Lypro Biosciences: Employment.

The mTOR inhibitor CCI-779 (temsirolimus) downregulates p21 and induces cell cycle arrest and autophagy in mantle cell lymphoma (MCL)

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 7573-7573 ◽  
Author(s):  
V. Y. Yazbeck ◽  
G. V. Georgakis ◽  
Y. Li ◽  
A. Younes
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Mantle Cell Lymphoma ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Antiproliferative Activity ◽  
Cell Lymphoma ◽  
Mtor Inhibition ◽  
Cycle Arrest ◽  
Mantle Cell

7573 Background: Mantle cell lymphoma (MCL) is a distinct type of B-cell lymphoma associated with transient response to conventional chemotherapy, continuous relapses and median survival of only 3–4 years. The mammalian target of rapamycin (mTOR) pathway is activated in many human malignancies where it regulates cyclin D1 translation. In a phase II trial, temsirolimus (CCI-779), an inhibitor of mTOR kinase used as single agent achieved an overall response rate of 38% in relapsed MCL patients. Our goal was to determine the activity and the mechanism of action of CCI-779 in MCL cell lines and to examine whether CCI-779 may synergizes with proteasome inhibitors. Methods: The activity of CCI-779 was determined in 3 mantle cell lymphoma cell lines (Jeko 1, Mino, Sp 53). Cell viability was determined by MTS assay, and autophagy by Acridine orange. Analysis of cell cycle was performed by flow cytometry and apoptosis by Annexin-V binding. Molecular changes were determined by western blot . Results: CCI-779 induced cell growth arrest in all cell lines in a time and dose dependent manner. The antiproliferative activity was due to cell cycle arrest in the G0/G1 phase followed by autophagy. CCI-779 decreased S6 phosphorylation in Jeko 1,Sp 53 indicative of mTOR inhibition. Furthermore, CCI-779 downregulated p21 expression in all three cell lines, without altering p 27 expression. Moreover, CCI-779 decreased the expression of the antiapoptotic protein cFLIP and ERK in both Jeko1 and Sp 53, but had no effect on cyclin D1 expression. The proteasome inhibitor bortezomib was also effective in all MCL cell lines, but failed to demonstrate significant synergy with CCI-779. Conclusions: The antiproliferative activity of CCI-779 in MCL is mediated by p21 downregulation and autophagy, without significant effect on cyclin D1 expression. The lack of synergy between bortezomib and CCI-779 should be confirmed using fresh MCL tumor cells. No significant financial relationships to disclose.

scholarly journals Abemaciclib-Based Combinational Therapies to Overcome Therapeutic Resistance in Mantle Cell Lymphoma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 33-34
Author(s):  
Yuxuan Che ◽  
Yang Liu ◽  
Lingzhi Li ◽  
Holly Hill ◽  
Joseph McIntosh ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Single Agent ◽  
Therapeutic Resistance ◽  
Cycle Arrest ◽  
Mantle Cell ◽  
Ic50 Values

Introduction The past decades witnessed dramatic improvement of overall survival rate of mantle cell lymphoma (MCL) patients by constant efforts in developing novel therapeutic strategies that include ibrutinib and venetoclax. Nevertheless, resistance is still a major challenge in refractory/relapsed MCL patients. Chromosomal translocation t(11:14)(q13:q32) of the cyclin D1 (CCND1) gene is the hallmark of MCL, which leads to overexpression of cyclin D1. This overexpression promotes aberrant cell cycle progression by activating CDK4/6. Abemaciclib is a selective CDK4/6 inhibitor used as a clinical treatment of breast cancer and has been shown to be effective in preclinical human MCL xenograft models. It has also been used in a phase II clinical trial as a single agent among refractory/relapsed MCL patients with an objective response rate of 35.7%. In this preclinical study, we aim to evaluate the benefit of a combinational therapeutic strategy using abemaciclib with other molecular targeting agents among MCL patients with therapeutic resistance. Methods Cytotoxic efficacy of abemaciclib as a single agent and in combination with other drugs on different MCL cell lines and primary lymphoma cells from MCL patients with or without resistance was used as a key criterion for screening beneficial therapeutic strategies. Cell apoptosis and cell cycle arrest assays were conducted to further evaluate those effective combinations. Western blot was performed to investigate the mechanism of action of the combinations. Finally, the efficacy of abemaciclib alone or in combination were assessed in ibrutinib-resistant or venetoclax-resistant MCL PDX models in vivo. Results Our preliminary data showed that all MCL cell lines involved in this study were highly sensitive to abemaciclib treatment with IC50 values ranging from 50 nM to 1 µM. Further investigation of abemaciclib cytotoxicity on ibrutinib and/or venetoclax resistant MCL cell lines showed effective inhibition with a higher IC50 values ranging from 5 µM to 10 µM. More importantly, abemaciclib had potent efficacy on cells from primary MCL patients as well as from patients with acquired ibrutinib resistance. Our recent findings revealed that the addition of PI3K inhibitor TGR-1202 significantly enhanced cytotoxicity of abemaciclib in both sensitive and resistant MCL cell lines. Abemaciclib significantly inhibited phosphorylation of Rb1, the active form of the protein, in 4 different MCL cell lines. The active Rb1 maintains the cell in the G1 phase, preventing progression through the cell cycle and acting as a growth suppressor. The result suggests that CDK4/6 inhibition with abemaciclib disrupts CDK4/6 suppressive activity towards pRb-E2F and induce cell cycle arrest in the MCL cells. Interestingly, abemaciclib somehow interrupted phosphorylation of Chk1, which is continuously phosphorylated and hence activated in the MCL cell lines. Inhibiting activation of Chk1 by abemaciclib may induce cell death via unmonitored and accumulated DNA damage. The efficacy of abemaciclib in combination with Bcl-2 or BTK inhibitors in MCL cell lines and isolated cells from MCL patients are ongoing. These data suggest that abemaciclib in combination with other therapeutic drugs could be beneficial in targeting therapeutic resistant MCL cells. Conclusions Abemaciclib showed impressive therapeutic potency on both MCL cell lines and isolated primary cells from MCL patients, which is likely due to the predominant contribution of cyclin D1-CDK4/6 pathway to malignancy. Other agents, such as PI3K inhibitors, can sensitize abemaciclib in therapeutic resistant MCL cells. Thus, an abemaciclib based multi-drug combinational strategy may be a promising therapy for refractory/relapsed MCL patients in the near future. Disclosures Wang: Beijing Medical Award Foundation: Honoraria; Lu Daopei Medical Group: Honoraria; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; Pulse Biosciences: Consultancy; Loxo Oncology: Consultancy, Research Funding; Targeted Oncology: Honoraria; OMI: Honoraria, Other: Travel, accommodation, expenses; Nobel Insights: Consultancy; Guidepoint Global: Consultancy; Dava Oncology: Honoraria; Verastem: Research Funding; Molecular Templates: Research Funding; OncLive: Honoraria; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Oncternal: Consultancy, Research Funding; Juno: Consultancy, Research Funding; BioInvent: Research Funding; VelosBio: Research Funding; Acerta Pharma: Research Funding; InnoCare: Consultancy; MoreHealth: Consultancy; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding.

Sign in / Sign up

Export Citation Format

Share Document