scholarly journals All trans retinoic acid nanodisks enhance retinoic acid receptor mediated apoptosis and cell cycle arrest in mantle cell lymphoma

2010 ◽  
pp. no-no ◽  
Author(s):  
Amareshwar T.K. Singh ◽  
Andrew M. Evens ◽  
Reilly J. Anderson ◽  
Jennifer A. Beckstead ◽  
Natesan Sankar ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Retinoic Acid ◽  
Mantle Cell Lymphoma ◽  
Cell Cycle Arrest ◽  
Cell Lymphoma ◽  
Retinoic Acid Receptor ◽  
Cycle Arrest ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Mantle Cell

All Trans Retinoic Acid Nanodisks Enhance Retinoic Acid Receptor-Mediated Apoptosis and Cell Cycle Arrest in Mantle Cell Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3722-3722
Author(s):  
Amareshwar Singh ◽  
Andrew M Evens ◽  
Reilly Anderson ◽  
Jennifer Beckstead ◽  
Natesan Sankar ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Retinoic Acid ◽  
Mantle Cell Lymphoma ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Retinoic Acid Receptor ◽  
Ros Generation ◽  
Cycle Arrest ◽  
Mantle Cell

Abstract Abstract 3722 Poster Board III-658 Background Mantle cell lymphoma (MCL) is characterized by the translocation t (11; 14)(q13; q32), aggressive clinical behavior, and poor patient outcomes following conventional chemotherapy. New treatment approaches are needed that target novel pathways. All-trans retinoic acid (ATRA) is a key retinoid that acts through nuclear receptors that function as ligand-inducible transcription factors. We hypothesized that since MCL cells express retinoid receptors, ATRA will exert anti-proliferative effects and thus may have a role in treatment. In the present study, we pursued a novel approach to deliver an ATRA payload to MCL cells in culture. This water insoluble bioactive lipid was stably incorporated into nanoscale lipid particles, termed nanodisks (ND). ND are comprised of a disk-shaped phospholipid bilayer whose periphery is stabilized by amphipathic apolipoproteins. It was hypothesized that following cellular uptake and/or liberation of ATRA from ND, this retinoid will interact with intracellular binding proteins and, ultimately, nuclear hormone receptors, leading to target gene transactivation, cell growth arrest and/or apoptosis. Methods We studied the mantle cell lymphoma cell lines Granta, NCEB and JEKO. ATRA-ND were prepared using recombinant human apolipoprotein A-I as the scaffold protein. Empty ND, lacking ATRA, were prepared in the same manner except that ATRA was omitted from the formulation contents. In various experiments ATRA was presented to cells using dimethylsulfoxide (DMSO) as vehicle (naked ATRA). Reactive oxygen species (ROS) were measured by oxidation of 2'7'dichlorofluorescein diacetate (H2DCFDA) to dichlorofluorescein (DCF) and quantified by fluorescence intensity using FACS. Apoptosis was quantified by Annexin V-FITC and PI using flow cytometry and FACS. Cell cycle analysis was measured by flow cytometry using FACS. PARP cleavage, caspase activation and analysis of cell cycle regulator proteins was measured by Western blotting, and retinoic acid receptor activity was measured by RT-PCR. Results We found that ATRA-ND induced significantly more cell death than naked ATRA (in dimethylsulfoxide) or empty ND. In all three cell lines, ATRA-ND induced reactive oxygen species (ROS) generation to a greater extent than naked ATRA. Incubation of cells with the antioxidant, N-acetylcysteine, inhibited ATRA-ND-induced apoptosis. Compared to naked ATRA, ATRA-ND enhanced G1 cell cycle arrest. Cyclin dependent kinases (CDK) regulate checkpoints that integrate mitogenic and growth inhibitory signals during cell cycle transitions. CDK inhibitors bind to CDK and inhibit kinase activity, leading to cell cycle arrest. P21 and p27 negatively regulate CDKs, and since p21 is induced by the tumor suppressor p53 in response to DNA damage, the expression level of these proteins was examined in Granta cells as a function of ATRA exposure. Compared to untreated control cells, no changes in the level of these proteins were seen following incubation with empty-ND. Whereas naked ATRA induced a modest increase in p21, p27 and p53, much larger increases were induced by ATRA-ND. Further ATRA-ND resulted in striking decrease in cyclin-D1. At ATRA concentrations that induce apoptosis, expression levels of the retinoic acid receptor- α (RAR α) and retinoid X receptor- γ (RXR γ) were increased. Furthermore, the RAR antagonist, Ro41-5253, inhibited ATRA-ND-induced ROS generation and abrogated ATRA-ND-induced cell growth arrest and apoptosis. Taken together, we found that ATRA-ND-induced apoptosis is dependent on ROS generation and RAR activation. We do not know if ATRA in ND is more stable or resists degradation during the course of cell incubations or whether ATRA-ND are taken up by the cells via receptor mediated endocytosis. Further work is required to elucidate the molecular basis of the enhanced biological activity of ATRA when presented to cells as a component of ND. Conclusion ATRA-ND greatly enhanced apoptosis and cell cycle arrest in MCL cell lines, and resulted in increase in p21, p27 and p53 with a decrease in cyclin D1. It is conceivable that solubilization of ATRA in the ND hydrophobic milieu effectively concentrates this bioactive lipid and provides a means for more efficient delivery to target cells. Nonetheless, results obtained in this study suggest ATRA-ND represent a potentially effective approach to the treatment of MCL and should be explored further. Disclosures: No relevant conflicts of interest to declare.

The HDACi Romidepsin Markedly Synergizes With Inhibition Of ATM By KU60019 In Mantle Cell Lymphoma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3066-3066 ◽  
Author(s):  
Luigi Scotto ◽  
Kelly Zullo ◽  
Xavier Jirau Serrano ◽  
Laura K Fogli ◽  
Owen A. O'Connor
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Mantle Cell Lymphoma ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Cdk Inhibitor ◽  
Protein Levels ◽  
Cycle Arrest ◽  
Mantle Cell

Abstract Mantle cell lymphoma (MCL) is a disease characterized by gross cell cycle dysregulation driven by the constitutive overexpression of cyclin D1. The identification of a “proliferation signature” in MCL, underscores the necessity of new therapeutic approaches aimed at lowering the proliferative signature of the disease, theoretically shifting the prognostic features of the disease. Romidepsin, an HDAC inhibitor (HDACi) approved for the treatment of relapsed T-cell lymphoma, is thought to induce cell cycle arrest and apoptosis. Central to the block of cell proliferation is the up-regulation of the cdk inhibitor p21Cip1/Waf1. However up-regulation of p21Cip1/Waf1 has also been shown to reduce sensitivity to romidepsin. HDACi activates p21Cip1/Waf1 expression via ATM and KU60019, a specific ATM inhibitor, has been shown to decrease the p21Cip1/Waf1 protein levels in a concentration dependent manner. We sought to explore the effect of the combination of romidepsin and KU60019 in inducing cell death in MCL. Analysis of romidepsin treated Jeko-1 cell extracts showed a marked effect on the expression of proteins involved in cell cycle regulation. Decrease expression of Emi1, a mitotic regulator required for the accumulation of the APC/C substrates was observed. Emi1 is also responsible for the stability of the E3 ubiquitin ligase Skp2 that specifically recognizes and promotes the degradation of phosphorylated cdk inhibitor p27. However, decrease in Emi1 protein levels, upon addition of romidepsin, was not followed by an increased expression of the cdk inhibitor p27. On the other end, increased expression of the cdk inhibitor p21Cip1/Waf1, was a common feature of all romidepsin treated MCL lines analyzed. Cell cycle analysis via Fluorescent Activated Cell Sorting (FACS) of romidepsin treated Jeko-1 cells showed an accumulation of romidepsin treated cells in the G2/M phase when compared to the control suggesting a p21Cip1/Waf1 induced cell cycle arrest. For all cytotoxicity assays, luminescent cell viability was performed using CellTiter-GloTM followed by acquisition on a Biotek Synergy HT and IC50s calculated using the Calcusyn software. Drug: drug interactions were analyzed using the calculation of the relative risk ratios (RRR). Synergy analyses were performed using Jeko-1, Maver-1 and Z-138 cells treated with different concentrations of romidepsin corresponding to IC10-20 in combination with KU60019 at a concentration of 2.5, 5.0, 7.5 and 15 umol/L for 24, 48 and 72 hours. A synergistic cytotoxic effect was observed in all MCL cell lines when the HDACi was combined with KU60019 throughout the range of all concentrations. The RRR analysis showed a strong synergism at 48 and 72 hours in virtually all combinations of HDACi and KU60019 in all three cell lines. The results of drug:drug combination in two of the three cell lines are shown below. Protein expression analysis of Jeko-1 and Maver-1cells treated with single agents or combinations for 48 hours revealed changes in a host of proteins known to be involved in cell cycle control and apoptosis. The increased p21 protein expression upon addition of romidepsin, was not observed when the romidepsin treatment was combined with the KU60019. Increased activation of the programmed cell death proteins Caspase 8, induced by Fas, and Caspase 3 was observed upon combinations of the single agents in all three cell lines, resulting in an increased cleavage of Poly (ADP-ribose) polymerase (PARP-1). Finally, the abundance of the anti-apoptotic proteins Bcl-XL and BCL-2 showed a significant decrease after treatment with romidepsin plus increase concentrations of KU60019 when compared with their abundance in the presence of the single agents. Cell cycle analysis of Jeko-1 cells treated for 24 hours with single agents and combination suggests that the increased apoptosis is the result of inhibition of the p21Cip1/Waf1 induced G2/M cell cycle arrest by KU60019. Overall, these data demonstrated that the combination of romidepsin and KU60019 was synergistically effective in inhibiting the in vitro growth of the mantle cell lymphoma lines. Jeko-1 Maver-1 Disclosures: O'Connor: Celgene: Consultancy, Research Funding.

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 In Vitro Anticancer Effects of All-trans Retinoic Acid in Combination with Dacarbazine against CD117+ Melanoma Cells

Drug Research ◽  
2020 ◽  
Vol 70 (12) ◽  
pp. 563-569
Author(s):  
Bahareh Mohammadi Jobani ◽  
Elham Mohebi ◽  
Nowruz Najafzadeh
Keyword(s):  
Cell Cycle ◽  
Retinoic Acid ◽  
Malignant Melanoma ◽  
Cell Cycle Arrest ◽  
Combined Treatment ◽  
Melanoma Cells ◽  
Cycle Arrest ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid

Abstract Background Malignant melanoma is a common form of skin cancer that contains different cell types recognized by various cell surface markers. Dacarbazine-based combination chemotherapy is frequently used for the treatment of melanoma. Despite its potent anticancer properties, resistance to dacarbazine develops in malignant melanoma. Here, we aim to improve response to dacarbazine therapy by pretreatment with all-trans retinoic acid (ATRA) in CD117+ melanoma cells. Methods The CD117+ melanoma cells were sorted from A375 malignant melanoma cell line using magnetic-activated cell sorting (MACS). The cell viability was examined by cell proliferation assay (MTT). Apoptosis was determined by acridine orange/ ethidium bromide staining. Indeed, we performed flow cytometry to evaluate the cell cycle arrest. Results Here, the CD117+ melanoma cells were incubated with various concentrations of ATRA, dacarbazine, and their combination to determine IC50 values. We found that 20 µM ATRA treatment followed by dacarbazine was found to be more effective than dacarbazine alone. There was an indication that the combination of ATRA with dacarbazine (ATRA/dacarbazine) caused more apoptosis and necrosis in the melanoma cells (P<0.05). Furthermore, ATRA/dacarbazine treatment inhibited the cell at the G0/G1 phase, while dacarbazine alone inhibited the cells at S phase. Conclusion Collectively, combined treatment with ATRA and dacarbazine induced more apoptosis and enhanced the cell cycle arrest of CD117+ melanoma cells. These results suggested that ATRA increased the sensitivity of melanoma cells to the effect of dacarbazine.

Molecular Mechanisms of the mTOR Inhibitor Temsirolimus (CCI-779) Antiproliferative Effects in Mantle Cell Lymphoma: Induction of Cell Cycle Arrest, Autophagy, and Synergy with Vorinostat (SAHA).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2493-2493 ◽  
Author(s):  
Victor Y. Yazbeck ◽  
Georgios V. Georgakis ◽  
Yang Li ◽  
Eiji Iwado ◽  
Seiji Kondo ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mantle Cell Lymphoma ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Mtor Inhibitor ◽  
Cell Lymphoma ◽  
Time Dependent ◽  
Dependent Manner ◽  
Cycle Arrest ◽  
Mantle Cell

Abstract Aberrant activation of the PI3-Kinase/Akt/mTOR survival pathway has been implicated in promoting the growth and survival of a variety of cancers, including lymphoma, and is currently being explored for cancer therapy. Importantly, the small molecule mTOR inhibitor temsirolimus (CCI-779) recently demonstrated significant clinical activity in patients with relapsed mantle cell lymphoma (MCL). However, the mechanism of action of temsirolimus in MCL cells is unknown. In this study, we demonstrated that temsirolimus induced cell growth inhibition in three MCL cell lines in a time-dependent and dose-dependent manner. The activity of temsirolimus was determined in 3 mantle cell lymphoma cell lines (Jeko-1, Mino, SP53). Temsirolimus upregulated p27 without altering cyclin D1 levels, resulting in cell cycle arrest in the G0/G1 phase. The Akt/mTOR pathway has been implicated in regulating cellular autophagy in yeasts and in mammalian cells. Thus, we examined whether temsirolimus may also induce autophagy in MCL cells, which is identified by the sequestering of cytoplasmic proteins into the lytic autophagosomes and autolysosome, and the formation of acidic vesicular organelles (AVOs). Temsirolimus induced AVOs formation indicative of autophagy in all MCL cell lines at doses ranging between 1 and 1000 nM in a time-dependent manner, with the highest activity observed between 72 and 96 hours of incubation. LC3 is essential for amino acid starvation-induced autophagy in yeasts. LC3-I is the cytoplasmic form, which is processed into the LC3-II form that is associated with the autophagosome membrane. Incubation of the SP53 cells with temsirolimus (1,000 nM) for 96 hours, resulted in processing LC3-I into LC3-II, indicative of autophagy induction. To further confirm induction of autophagy, SP53 cells expressing LC3-fused green fluorescent protein (GFP-LC3) were treated with temsirolimus and the pattern of LC3 distribution was compared with untreated cells using fluorescence microscopy. Untreated control cells showed a diffuse cytoplasmic distribution of LC3, whereas temsirolimus -treated cells showed a punctate pattern of green fluorescence, indicative of its association with autophagosomes. Furthermore, temsirolimus increased acidic vesicular organelles and microtubule-associated protein 1 light chain 3 (LC3) processing as determined by Western blot, which are characteristic of autophagy. In contrast, temsirolimus had minimal induction of apoptosis. Moreover, temsirolimus inhibited ribosomal S6 phosphorylation, an mTOR downstream target. The histone deacetylase inhibitor vorinostat (suberoylanilide hydroxamic acid, SAHA) demonstrated antiproliferative activity in a dose and time dependent manner in all three MCL cell lines. SAHA enhanced the activity of temsirolimus, which was associated with ERK dephosphorylation and caspase 3 activation. In contrast, temsirolimus did not potentiate the antitumor effects of bortezomib, doxorubicin, or gemcitabine. Our results demonstrate that in short-term culture, temsirolimus is primarily a cytostatic drug, and suggest that SAHA may potentiate the clinical efficacy of temsirolimus patients with MCL.

scholarly journals RNAi screen identifies UBE2D3 as a mediator of all-trans retinoic acid-induced cell growth arrest in human acute promyelocytic NB4 cells

Blood ◽  
2007 ◽  
Vol 110 (2) ◽  
pp. 640-650 ◽  
Author(s):  
Hidenori Hattori ◽  
Xueqing Zhang ◽  
Yonghui Jia ◽  
Kulandayan K. Subramanian ◽  
Hakryul Jo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Retinoic Acid ◽  
Cell Growth ◽  
Cyclin D1 ◽  
Cell Cycle Arrest ◽  
Growth Arrest ◽  
Cycle Arrest ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid

Abstract All-trans retinoic acid (ATRA) has been widely used in differentiation therapy for acute promyelocytic leukemia (APL). ATRA binds to retinoic acid receptor (RAR) and triggers the formation of the transcription coactivator complex, which leads to changes in gene expression, APL cell-cycle arrest and differentiation, and clinical remission. The mechanisms responsible for ATRA's beneficial effects are still ill-defined. Here, we conducted a large-scale, unbiased short hairpin RNA (shRNA) screen aiming to identify mediators of ATRA-induced differentiation and growth arrest of APL cells. Twenty-six proteins were identified. They cover a wide range of cellular functions, including gene expression, intracellular signaling, cell death control, stress responses, and metabolic regulation, indicating the complexity of ATRA-induced cell growth control and differentiation in APL. One of these proteins, the ubiquitin-conjugating enzyme UBE2D3, is up-regulated in ATRA-treated acute promyelocytic NB4 cells. UBE2D3 is physically associated with cyclin D1 and mediates ATRA-induced cyclin D1 degradation. Knocking down UBE2D3 by RNA interference (RNAi) leads to blockage of ATRA-induced cyclin D1 degradation and cell-cycle arrest. Thus, our results highlight the involvement of the ubiquitin-mediated proteolysis pathway in ATRA-induced cell-cycle arrest and provide a novel strategy for modulating ATRA-elicited cellular effects.

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