scholarly journals Autophagy and Ubiquitin-Mediated Proteolytic Degradation of PML/Rarα Fusion Protein in Matrine-Induced Differentiation Sensitivity Recovery of ATRA-Resistant APL (NB4-LR1) Cells: In Vitro and In Vivo studies

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2646-2646
Author(s):  
Dijiong Wu ◽  
Keding Shao ◽  
Qihao Zhou ◽  
Jie Sun ◽  
Ziqi Wang ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Fusion Protein ◽  
Protein Degradation ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Conflicts Of Interest ◽  
Promyelocytic Leukemia ◽  
Nb4 Cells ◽  
Ubiquitin Proteasome

Abstract The major feature that distinguishes acute promyelocytic leukemia (APL) cells from other malignant hematopoietic cells is the expression of promyelocytic leukemia-retinoicacid receptor α (PML-RARα) fusion protein, which contributes to the inhibition of RARα-regulated hematopoietic cellular differentiation. The complete remission rate of APL exceeded 90% with the application of all-trans retinoic acid (ATRA), arsenic trioxide (ATO), and anthracycline-based chemotherapy. However, the 7-year cumulative incidence of relapses was reported as 28.6% in APL maintenance with ATRA and daunorubicin and even reached 33% in the ATRA maintenance treatment group. The relapse/refractory patients showed resistance to ATRA and/or ATO, which has been identified as a clinically significant problem. Currently, no effective drugs are available to reverse the ATRA resistance. Matrine (MAT) is the main active component of Sophora flavescens. It has been used to treat chronic hepatitis for several years in China. Recently, the molecule has been proven to exhibit an anti-leukemic effect. Previous studies have revealed that MAT can reverse the ATRA resistance of NB4-LR1 cells when coupled with ATRA. The treatment with 0.1mmol/L MAT and 1μmol/L ATRA can restore the ability of NB4-LR1 cells to differentiate, which might be related to the increased level of cyclic adenosine monophosphate and protein kinase A activity in NB4-LR1 cells, reduced telomerase activity and downregulated expression of topoisomerase II beta (TopoIIβ) (Wu, et al. Planta Med 2014). Subsequently, this study aimed to investigate the mechanism underlying the degradation of the PML/RARα fusion protein in the presence of MAT and ATRA in NB4 and NB4-LR1 cell lines. ATRA-sensitive (NB4) and ATRA-resistant (NB4-LR1) cell lines were used. Nitroblue tetrazolium reduction assay and flow cytometry were used to detect the differentiation ability. The activity of ubiquitin-proteasome and autophagy-mediated pathways in both cells treated with ATRA with or without MAT were compared in protein and mRNA level (Western blot analysis, qRT-PCR), the Fluorescent substrate Suc-LLVY-AMC detection was used to detect the activity of proteasome, and electron microscope for observing autophagosome. MG 132(proteasome inhibitor), rapamycin (autophagy activator, RAPA), hydroxychloroquine (lysosomal inhibitor, HCQ) and STI571 [retinoic acid receptor alpha (RARα) ubiquitin stabilizer] were used as positive controls. The effect of MAT was observed in vivo using xenografts. Results showed that MAT improved the sensitivity of NB4-LR1cells to ATRA treatment, which was consistent with the expression of PML-RARα fusion protein (Fig.A-C).The ubiquitin proteasome pathway plays a crucial role in protein degradation. MAT promoted the ubiquitylation level in NB4-LR1 by stabilized the 20S protein expression and enhanced the activity of the proteasome (Fig.D). ATRA inhibited the expression of RARα in NB4-LR1 cells, which was contradictory to that in NB4 cells. MAT can stabilize the expression of RARα in NB4-LR1 cells, whereas MG132 downregulated the expression of RARα in both cell lines, which hampered the differentiation of NB4 cells (Fig.E-G). In addition to UPP, the autophagy pathway also had a significant role in arsenious acid- or ATRA-mediated PML-RARα fusion protein degradation. MAT could promoted the autophagy in NB4-LR1 cells, with an increase in microtubule-associated protein 1 light chain3 (LC3)-II and LC3-II/LC3-I ratio and exhaustion of P62 (Fig.H-K). A similar phenomenon was observed in mouse xenografts (Fig.L-N). In summary, the present study revealed the difference in the chain reaction of sensitive and resistant APL cell lines (NB4 and NB4-LR1, respectively) to the treatment of ATRA, explaining the mechanism underlying the resistance to ATRA. ATRA decrease the level of 20S core subunit and the RARα in NB4-LR1 cells, but could not activate the autophagy process. These effects were reversed by the combination of MAT. The proteasome inhibitor might hamper the RARα stabilization and hence was not advantageous for the differentiation of cells. It also induced autophagy in NB4-LR1 cells. MAT induced the activation of UPP and mediated the autophagic degradation process, which synergistically induced the degradation of PML-RARα fusion protein and promote the differentiation of NB4-LR1 cells. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Trivalent Antimonials Induce Degradation of the PML-RAR Oncoprotein and Reorganization of the Promyelocytic Leukemia Nuclear Bodies in Acute Promyelocytic Leukemia NB4 Cells

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4308-4316 ◽  
Author(s):  
Stefan Müller ◽  
Wilson H. Miller ◽  
Anne Dejean
Keyword(s):  
Retinoic Acid ◽  
Fusion Protein ◽  
Acute Promyelocytic Leukemia ◽  
Promyelocytic Leukemia ◽  
Nuclear Bodies ◽  
Antimony Trioxide ◽  
Nb4 Cells ◽  
Pml Nuclear Bodies

Acute promyelocytic leukemia (APL) is characterized by a specific t(15;17) chromosomal translocation that fuses the genes encoding the promyelocytic leukemia protein (PML) and the retinoic acid receptor  (RAR). The resulting PML-RAR protein induces a block in the differentiation of the myeloid progenitor cells, which can be released by retinoic acid (RA) in vitro and in vivo. The RA-induced differentiation of APL blasts is paralleled by the degradation of the fusion protein and the relocation of wild-type PML from aberrant nuclear structures to its normal localization in nuclear bodies. Recently, arsenic trioxide (As2O3) treatment was proposed as an alternative therapy in APL, because it can induce complete remission in both RA-sensitive and -resistant APL patients. Intriguingly, As2O3 was also shown to induce degradation of the PML-RAR chimera and to reorganize PML nuclear bodies. Here we show that trivalent antimonials also have striking effects on RA-sensitive and RA-resistant APL cells. Treatment of the APL-derived NB4 cells and the RA-resistant subclone NB4R4 with antimony trioxide or potassium antimonyl tartrat triggers the degradation of the fusion protein and the concomitant reorganization of the PML nuclear bodies. In addition, as reported for As2O3, the antimonials provoke apoptosis of NB4 and NB4R4 cells. The mechanism of antimony action is likely to be similar to that of As2O3, notably both substances induce the attachment of the ubiquitin-like SUMO-1 molecule to the PML moiety of PML-RAR. From these data, we propose that, in analogy to As2O3, antimonials might have a beneficial therapeutic effect on APL patients, perhaps with less toxicity than arsenic.

Trivalent Antimonials Induce Degradation of the PML-RAR Oncoprotein and Reorganization of the Promyelocytic Leukemia Nuclear Bodies in Acute Promyelocytic Leukemia NB4 Cells

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4308-4316 ◽  
Author(s):  
Stefan Müller ◽  
Wilson H. Miller ◽  
Anne Dejean
Keyword(s):  
Retinoic Acid ◽  
Fusion Protein ◽  
Acute Promyelocytic Leukemia ◽  
Promyelocytic Leukemia ◽  
Nuclear Bodies ◽  
Antimony Trioxide ◽  
Nb4 Cells ◽  
Pml Nuclear Bodies

Abstract Acute promyelocytic leukemia (APL) is characterized by a specific t(15;17) chromosomal translocation that fuses the genes encoding the promyelocytic leukemia protein (PML) and the retinoic acid receptor  (RAR). The resulting PML-RAR protein induces a block in the differentiation of the myeloid progenitor cells, which can be released by retinoic acid (RA) in vitro and in vivo. The RA-induced differentiation of APL blasts is paralleled by the degradation of the fusion protein and the relocation of wild-type PML from aberrant nuclear structures to its normal localization in nuclear bodies. Recently, arsenic trioxide (As2O3) treatment was proposed as an alternative therapy in APL, because it can induce complete remission in both RA-sensitive and -resistant APL patients. Intriguingly, As2O3 was also shown to induce degradation of the PML-RAR chimera and to reorganize PML nuclear bodies. Here we show that trivalent antimonials also have striking effects on RA-sensitive and RA-resistant APL cells. Treatment of the APL-derived NB4 cells and the RA-resistant subclone NB4R4 with antimony trioxide or potassium antimonyl tartrat triggers the degradation of the fusion protein and the concomitant reorganization of the PML nuclear bodies. In addition, as reported for As2O3, the antimonials provoke apoptosis of NB4 and NB4R4 cells. The mechanism of antimony action is likely to be similar to that of As2O3, notably both substances induce the attachment of the ubiquitin-like SUMO-1 molecule to the PML moiety of PML-RAR. From these data, we propose that, in analogy to As2O3, antimonials might have a beneficial therapeutic effect on APL patients, perhaps with less toxicity than arsenic.

scholarly journals Autophagy and Ubiquitin-Mediated Proteolytic Degradation of PML/Rarα Fusion Protein in Matrine-Induced Differentiation Sensitivity Recovery of ATRA-Resistant APL (NB4-LR1) Cells: in Vitro and in Vivo Studies

2018 ◽  
Vol 48 (6) ◽  
pp. 2286-2301 ◽  
Author(s):  
Dijiong  Wu ◽  
Keding Shao ◽  
Qihao Zhou ◽  
Jie Sun ◽  
Ziqi Wang ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Fusion Protein ◽  
Cell Lines ◽  
Mrna Level ◽  
In Vivo Studies ◽  
Proteolytic Degradation ◽  
Induced Differentiation ◽  
Fluorescent Substrate

Background/Aims: Although the cure rate of acute promyelocytic leukemia (APL) has exceeded 90%, the relapse/refractory APL that resistant to all-trans retinoic acid (ATRA) or ATO was still serious concern. Matrine (MAT) could improve the differentiation ability of ATRA-resistant APL cells. This study aimed to explore how the APL-specific fusion protein was degraded in ATRA-resistant APL with the application of MAT and ATRA. Methods: ATRA-sensitive (NB4) and ATRA-resistant (NB4-LR1) cell lines were used. Nitroblue tetrazolium reduction assay and flow cytometry were used to detect the differentiation ability. The activity of ubiquitin-proteasome and autophagy-mediated pathways in both cells treated with ATRA with or without MAT were compared in protein and mRNA level (Western blot analysis, qRT-PCR), the Fluorescent substrate Suc-LLVY-AMC detection was used to detect the activity of proteasome, and electron microscope for observing autophagosome. MG 132(proteasome inhibitor), rapamycin (autophagy activator), hydroxychloroquine (lysosomal inhibitor) and STI571 [retinoic acid receptor alpha (RARα) ubiquitin stabilizer] were used as positive controls. The effect of MAT was observed in vivo using xenografts. Results: MAT improved the sensitivity of NB4-LR1cells to ATRA treatment, which was consistent with the expression of PML-RARα fusion protein. MAT promoted the ubiquitylation level in NB4-LR1. MG 132 induced the decrease in RARα in both cell lines, and hampered the differentiation of NB4 cells. MAT also promoted the autophagy in NB4-LR1 cells, with an increase in microtubule-associated protein 1 light chain3 (LC3)-II and LC3-II/LC3-I ratio and exhaustion of P62. The expression of LC3II increased significantly in the MAT and ATRA + MAT groups in combination with lysosomal inhibitors. A similar phenomenon was observed in mouse xenografts. MAT induced apoptosis and differentiation. Conclusions: Autophagy and ubiquitin-mediated proteolytic degradation of PML/RARα fusion protein are crucial in MAT-induced differentiation sensitivity recovery of NB4-LR1 cells.

scholarly journals In vivo analysis of the role of aberrant histone deacetylase recruitment and RARα blockade in the pathogenesis of acute promyelocytic leukemia

2006 ◽  
Vol 203 (4) ◽  
pp. 821-828 ◽  
Author(s):  
Hiromichi Matsushita ◽  
Pier Paolo Scaglioni ◽  
Mantu Bhaumik ◽  
Eduardo M. Rego ◽  
Lu Fan Cai ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Fusion Protein ◽  
Acute Promyelocytic Leukemia ◽  
Histone Deacetylases ◽  
Retinoic Acid Receptor ◽  
Promyelocytic Leukemia ◽  
Dominant Negative

The promyelocytic leukemia–retinoic acid receptor α (PML-RARα) protein of acute promyelocytic leukemia (APL) is oncogenic in vivo. It has been hypothesized that the ability of PML-RARα to inhibit RARα function through PML-dependent aberrant recruitment of histone deacetylases (HDACs) and chromatin remodeling is the key initiating event for leukemogenesis. To elucidate the role of HDAC in this process, we have generated HDAC1–RARα fusion proteins and tested their activity and oncogenicity in vitro and in vivo in transgenic mice (TM). In parallel, we studied the in vivo leukemogenic potential of dominant negative (DN) and truncated RARα mutants, as well as that of PML-RARα mutants that are insensitive to retinoic acid. Surprisingly, although HDAC1-RARα did act as a bona fide DN RARα mutant in cellular in vitro and in cell culture, this fusion protein, as well as other DN RARα mutants, did not cause a block in myeloid differentiation in vivo in TM and were not leukemogenic. Comparative analysis of these TM and of TM/PML−/− and p53−/− compound mutants lends support to a model by which the RARα and PML blockade is necessary, but not sufficient, for leukemogenesis and the PML domain of the fusion protein provides unique functions that are required for leukemia initiation.

Anti-CD138-IFNα Fusion Proteins Are Effective in Treating Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 939-939
Author(s):  
Esther Yoo ◽  
Alex Vasuthasawat ◽  
Danh Tran ◽  
Alan Lichtenstein ◽  
Sherie Morrison
Keyword(s):  
Multiple Myeloma ◽  
Fusion Protein ◽  
Cell Lines ◽  
Dna Content ◽  
Fusion Proteins ◽  
Conflicts Of Interest ◽  
Myeloma Cell ◽  
Inhibition Of Proliferation ◽  
Discontinue Therapy

Abstract Abstract 939 Although IFNα has shown some efficacy in the treatment of multiple myeloma (MM), this efficacy has been limited in large part because systemic toxicity makes it difficult if not impossible to reach therapeutically effective doses at the site of the tumor. The short half-life of IFN also makes it difficult to sustain high levels during treatment, and because of the side effects, the patients often discontinue therapy. To address these issues, we have genetically fused IFNα2 to a chimeric IgG1 antibody specific for the antigen CD138 expressed on the surface of MM cells, yielding anti-CD138-IFNα. We have also produced a fusion protein (anti-CD138-mutIFNα) using a mutant IFNα that binds the IFN receptor (IFNAR) more tightly. The fusion proteins continued to bind CD138 and retained IFN activity and showed anti-proliferative activity against a broad panel of myeloma cell lines (HMCL) representing MM with different characteristic. To investigate the events responsible for the inhibition of proliferation, 8226/S, ANBL-6, MM1-144, H929, OCI-My5 and U266 cells were incubated with 500 pM anti-CD138-IFNα for 72 h and their DNA content analyzed by FLOW cytometry following permeabilization and staining with PI. The different cell lines exhibited different responses. All of the cell lines except OCI-My5 underwent apoptosis. For 8226/S, OCI-My5 and U266 there was little change in DNA content following treatment. ANBL-6 showed a slight increase in the number of cells in S. However, MM1-144 and H929 showed a marked accumulation in G2 with H929 also showing accumulation of cells with sub-G0content of DNA. Therefore, there is heterogeneity in the response of different HMCL to treatment with targeted IFNα2. For many but not all of the cell lines, anti-CD138-mutIFNα was more effective than anti-CD138-IFNα in inhibiting proliferation and causing DNA fragmentation. Anti-CD138-mutIFNα was more effective than anti-CD138-IFNα in inducing senescence-associated β-galactosidase and STAT1 activation in OCI-My5 cells. Treatment with anti-CD138-IFNα or anti-CD138-mutIFNα resulted in a decrease in the amount of IRF4 present in U266, suggesting that this may be responsible for the efficacy of the fusion proteins in this cell line. Treatment of the other cell lines did not alter the level of IRF4 present, but anti-CD138-IFNα and anti-CD138-mutIFNα treatment caused a decrease in the amount of ppRB present in 8226/S, OCI-My5 and MM1-144, and to a lesser extent in H929. To determine the in vivo efficacy of fusion protein treatment, SCID mice were injected subcutaneously with OCI-My5 cells and treated intravenously on days 14, 16 and 18 with 100 μg of the indicated proteins and monitored for tumor growth (Figure 1). Mice were sacrificed when tumors exceeded 1.5 cm in diameter. Treatment with anti-CD138-IFNα provided some protection (p ≤ 0.0001 compared to PBS). However, treatment with anti-CD138-mutIFNα was even more effective (p = 0.0004 compared to anti-CD138-IFNα). Anti-CD138-mutIFNα was also found to be more effective than anti-CD138-IFNα against primary MM cells. Patients with active myeloma were biopsied while off therapy and the marrow cells isolated by a negative antibody selection to >95% purity. After 72 h incubation with 25 nM of protein, anti-CD138 was found to have little effect. In contrast treatment with anti-CD138-IFNα caused a decrease in viability with anti-CD138-mutIFNα treatment leading to an even greater decrease in cell viability. Following 72 h of treatment, 25 nM of anti-CD138-mutIFNα was found to have more potent cytoreductive effects than 100 nM of anti-CD138-IFNα. Disclosures: No relevant conflicts of interest to declare.

Metabolic Rewiring Drives Resistance to Arsenic Trioxide in Acute Promyelocytic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3956-3956 ◽  
Author(s):  
Nithya Balasundaram ◽  
Saravanan Ganesan ◽  
Hamenth Kumar Palani ◽  
Ansu Abu Alex ◽  
Sachin David ◽  
...  
Keyword(s):  
Glucose Uptake ◽  
Acute Promyelocytic Leukemia ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Resistant Cell ◽  
Promyelocytic Leukemia ◽  
Leukemic Cells ◽  
Synergistic Activity ◽  
Normal Peripheral Blood ◽  
Nb4 Cells

Abstract The focus of ATO resistance in acute promyelocytic leukemia (APL) has centered on mutations in PML-RARA gene (Blood 2011, NEJM 2014). However such mutations are rare and cannot explain the majority of relapses seen in the clinic. To evaluate the mechanisms of ATO resistance, we generated ATO resistant NB4 sub clone NB4-EVAsR1 (A216V - VAF-91.7%) in our laboratory. We also had another ATO resistant cell line (UF1) which does not have the A216V mutation. In an expression array we noted that redox signaling, AMPK signaling and energy metabolism pathways were significantly dysregulated in the ATO resistant cell lines compared to naïve NB4 cells. Towards validating the microarray data and to characterize the ATO resistant cell lines we measured the basal levels of reactive oxygen species (ROS), glutathione(GSH), mitochondrial membrane potential (MMP), glucose uptake and their sensitivity to glycolytic inhibitor 2-Deoxy glucose (2-DG) in comparison to naïve NB4 cells. We observed that resistant cell lines have significantly lower ROS, MMP, glucose uptake (Fig 1a) and increased GSH. We also observed that the resistant cell lines were significantly less susceptible to treatment with 2-DG in comparison to naïve NB4 cells (Fig 1b) suggesting that resistant cell lines were less dependent on glycolysis. ATO has been reported to directly inhibit the glycolytic pathway, this effect is believed to contribute to its cytotoxic effect (PNAS 2015). However, we did not observe any cytotoxic synergy between ATO and 2-DG on naïve NB4 cells and neither did this combination restore sensitivity to ATO in the resistant cell lines (Fig 1b). Next we assessed the sensitivity of these resistant cell lines to oxidative phosphorylation (OXPHOS) inhibitors. We used an uncoupler (FCCP at 10uM) of OXPHOS which promotes uncoupled respiration by deregulating the proton gradient which drives ATP synthesis via ATP synthase. We observed that the FCCP treatment alone did not reduced the viability of naïve NB4 cells. Similarly, viability of ATO resistant cell lines also did not reduce significantly suggesting the ability of these cells to uncouple their metabolic pathway from OXPHOS to glycolysis when inhibited. However, when FCCP was combined with ATO it significantly restored the sensitivity of the resistant cell lines to ATO (Fig 1c). The same combination did not have any additive effect on naïve NB4 cells. The combination not only restored the sensitivity of the ATO resistant cell lines but also sensitized the conventionally ATO resistant cell lines such U937 (Fig 1c) and THP1. In spite of the profound effect on leukemic cells we also observed a significant bystander effect on the normal peripheral blood mononuclear cells (Fig 1c). The data suggests that the sensitivity of these resistant cell lines could be potentially restored by combining ATO with an OXPHOS uncoupler. A number of molecules that are FDA approved and used in the clinic also have OXPHOS uncoupling activity and could potentially be evaluated for their synergistic activity with ATO in leukemia. This data also draws attention to possible severe systemic off-target toxicity of such combinations which may be inadvertently used in the clinic. Disclosures No relevant conflicts of interest to declare.

scholarly journals Heat Shock Protein 70 Inhibitor, Alone or in Combination with Bortezomib, Prevented Plasmacytoma Development in Immunodeficient Mice Transplanted with Myeloma Cell Lines

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5658-5658
Author(s):  
Mariana Bleker de Oliveira ◽  
Angela Isabel Eugenio ◽  
Veruska Lia Fook Alves ◽  
Daniela Zanatta ◽  
Mihoko Yamamoto ◽  
...  
Keyword(s):  
Cell Line ◽  
Tumor Cells ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Conflicts Of Interest ◽  
Control Group ◽  
Immunodeficient Mice ◽  
Late Apoptosis

Abstract Introduction: HSP70 has an integrative role in protein degradation due to the interaction with many pathways, such as ubiquitin proteasome (UPS), unfolded protein response (UPR) and autophagy. In multiple myeloma (MM) HSP70 is overexpressed and helps to prevent proteotoxic stress and cell death caused by overload of unfolded/misfolded proteins produced by tumor cells. Aims: To explore the role of HSP70 inhibition, isolated or in association with proteasome inhibitor, as therapeutic strategy for MM through in vitro and in vivo analyses. Methods: RPMI8226-LUC-PURO and U266-LUC-PURO bioluminescent cell lines were treated with HSP70 inhibitor (VER155008- 50 μM or 80μM) and proteasome inhibitor (bortezomib 100nM) for evaluation of apoptosis induction by flow cytometry using annexin V and propidium iodide. NOD.Cg-rkdcscid Il2rgtm1Wjl/SzJ immunodeficient mice were used for plasmacytoma xenograft model and treated with intravenous VER155008 (40mg/kg) and bortezomib (1mg/kg), immediately after transplant of RPMI8226-LUC-PURO and U266-LUC-PURO bioluminescent cell lines (N=3 for each group, including controls, bortezomib, VER155008, and combination of bortezomib and VER155008). Bioluminescence was measured in IVIS Kinetic (Capiler Life Science) once a day for seven days. Results: Bortezomib used as single treatment was able to induce apoptosis in RPMI8226-LUC-PURO cell line: the best result for in vitro studies RPMI8226-LUC-PURO was 65% of late apoptosis after treatment with bortezomib. On the other hand, U266-LUC-PURO cell line presented higher percentage of apoptosis when treated with bortezomib and VER155008 combination: U266-LUC-PURO cell line presented more than 60% of late apoptosis after VER155008 (80μM) combined with bortezomib, showing that inhibition of HSP70 could overcome U266-LUC-PURO resistance to bortezomib alone. Mice treated with VER155008, alone or in combination with bortezomib, showed complete inhibition of tumor growth (absence of bioluminescence) for both cell lines when compared with control group after one week of treatment (p<0.001, Two-way ANOVA). Therefore, in vivo studies using mice treated with VER155008, alone or in combination with bortezomib, prevented tumor development after one week of treatment, independent of the cell line used in the xenotransplant. Conclusion: Our study shows that HSP70 and proteasome inhibitors combination induced apoptosis in tumor cells in vivo for both MM cell lines. Since HSP70 is overexpressed in MM and connects several signaling pathways that maintain cell survival, such as UPS, UPR and autophagy, it can represent a key role to establish a new approach for the treatment of MM. Financial support: FAPESP 2010/17668-6 and CNPq (155272/2013-6). UNIFESP Ethics Committee (0219/12). Disclosures No relevant conflicts of interest to declare.

Combined effect of all-trans retinoic acid and arsenic trioxide in acute promyelocytic leukemia cells in vitro and in vivo

Blood ◽  
2001 ◽  
Vol 97 (1) ◽  
pp. 264-269 ◽  
Author(s):  
Yongkui Jing ◽  
Long Wang ◽  
Lijuan Xia ◽  
Guo-qiang Chen ◽  
Zhu Chen ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Arsenic Trioxide ◽  
Acute Promyelocytic Leukemia ◽  
Promyelocytic Leukemia ◽  
Nb4 Cells ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Induced Apoptosis

Abstract All-trans retinoic acid (tRA) and arsenic trioxide (As2O3) induce non–cross-resistant complete clinical remission in patients with acute promyelocytic leukemia with t(15;17) translocation and target PML-RARα, the leukemogenic protein, by different pathways suggesting a possible therapeutic synergism. To evaluate this possibility, this study examined the effect of As2O3 on tRA-induced differentiation and, conversely, the effect of tRA on As2O3-induced apoptosis. As2O3 at subapoptotic concentrations (0.5 μM) decreased tRA-induced differentiation in NB4 cells but synergized with atRA to induce differentiation in tRA-resistant NB4 subclones MR-2 and R4 cells as measured by nitroblue tetrazolium reduction and tRA-inducible genes (TTGII, RARβ, RIG-E). tRA cleaved PML-RARα into distinct fragments in NB4 but not in tRA-resistant MR-2 or R4 cells, whereas As2O3 completely degraded PML-RARα in all 3 cell lines. As2O3-induced apoptosis was decreased by tRA pretreatment of NB4 cells but not of R4 cells and was associated with a strong induction of Bfl-1/A1 expression, a Bcl-2 protein family member. Severe combined immunodeficient mice bearing NB4 cells showed an additive survival effect after sequential treatment, but a toxic effect was observed after simultaneous treatment with tRA and As2O3. These data suggest that combined As2O3 and tRA treatment may be more effective than single agents in tRA-resistant patients. Although in vitro data do not always translate to in vivo response, toxicity and potential drug antagonism may be diminished by decreasing the concentration of As2O3 when given at the same time with therapeutic levels of tRA.

scholarly journals All-trans retinoic acid upregulates thrombomodulin and downregulates tissue-factor expression in acute promyelocytic leukemia cells: distinct expression of thrombomodulin and tissue factor in human leukemic cells

Blood ◽  
1994 ◽  
Vol 84 (9) ◽  
pp. 3001-3009 ◽  
Author(s):  
T Koyama ◽  
S Hirosawa ◽  
N Kawamata ◽  
S Tohda ◽  
N Aoki
Keyword(s):  
Retinoic Acid ◽  
Tissue Factor ◽  
Acute Promyelocytic Leukemia ◽  
Cell Lines ◽  
Promyelocytic Leukemia ◽  
Leukemic Cells ◽  
Monocytic Leukemia ◽  
Nb4 Cells ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid

The expressions of thrombomodulin (TM) and tissue factor (TF) by all- trans retinoic acid (ATRA) were studied in human leukemic cell lines including NB4 (acute promyelocytic leukemia) and U937 (monoblastic leukemia). ATRA remarkably upregulated TM antigen expression in cell lysates as well as TM cofactor activity on the cell surfaces of NB4. The level of TM mRNA in NB4 cells was increased by ATRA. Inherently procoagulant NB4 cells contained markedly higher content of TF, which was efficiently reduced by ATRA. Modest increase of TM and decrease of TF were observed when NB4 cells were treated with dibutyryl cyclic adenosine monophosphate (dbcAMP). On the other hand, both ATRA and dbcAMP showed dramatic increase of TM antigen level and modest decrease of TF antigen in U937 cells. These results suggest that ATRA regulates expressions of TM and TF antigens and activity in NB4 and U937 cell lines, and provide evidence for a potential efficiency of ATRA as a preventive and therapeutic agent for disseminated intravascular coagulation in promyelocytic and monocytic leukemia.

C/EBP Is Deregulated by PML-RAR in Acute Promyelocytic Leukemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3015-3015
Author(s):  
Florence Guibal ◽  
Hanna S. Radomska ◽  
Lisa M. Johansen ◽  
Daniel G. Tenen
Keyword(s):  
Retinoic Acid ◽  
Fusion Protein ◽  
Acute Promyelocytic Leukemia ◽  
Promyelocytic Leukemia ◽  
Myeloid Differentiation ◽  
Computer Search ◽  
Granulocytic Differentiation ◽  
Transient Transfection Assay

Abstract Acute promyelocytic leukemia (APL) cells are blocked at the promyelocyte stage of myeloid differentiation. The majority of APL cells display the t(15;17) reciprocal chromosomal translocation leading to the expression of the fusion protein promyelocytic leukemia-retinoic acid receptor alpha (PML-RARa). Cells harboring this reciprocal translocation can be induced to differentiate after treatment with all-trans retinoic acid (at-RA) both in vivo and in vitro. During normal hematopoiesis, differentiation is regulated by several key transcription factors. One of them, CCAAT/enhancer binding protein alpha (C/EBPa), controls expression of genes regulating normal myeloid differentiation. Its disruption leads to a block of granulocytic differentiation. We thus hypothesize that C/EBPa could be deregulated in APL and therefore participate in the pathogenesis of APL. Using the U937PR9 cell line, which expresses an inducible PML-RARa, we observed that expression of PML-RARa induced a decrease of both C/EBPa mRNA and protein, leading to decreased C/EBPa DNA binding activity. Using a transient transfection assay with a C/EBPa promoter construct in presence or absence of PML-RARa, we are able to demonstrate that PML-RARa can repress C/EBPa promoter activity. This repression is specific to the fusion protein, as both PML and RARa have no effect upon the C/EBPa promoter. A computer search of the C/EBPa promoter sequence did not exhibit any evident RARE binding site, and therefore we are currently mapping the site(s) responsible for this repression. In conclusion, PML-RARa down regulates C/EBPa expression; this down regulation could participate in the pathogenesis of APL. This hypothesis is also supported by the observation that at-RA treatment of APL cell lines (NB4 and HT93) induces a rapid restoration of both C/EBPa RNA and protein. Thus, a decrease in both C/EBPa expression and activity could contribute to the differentiation block of APL cells by deregulating the normal myeloid differentiation program.

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