Curcumin Enhances Differentiation of All-Trans Retinoic Acid (ATRA)-Sensitive and ATRA-Resistant Acute Promyelocytic (APL) Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4456-4456 ◽  
Author(s):  
Ameet R. Kini ◽  
Moolky Nagabhushan ◽  
Martin S. Tallman ◽  
Shantanu Roychowdhury
Keyword(s):  
Retinoic Acid ◽  
Therapeutic Agent ◽  
Atra Treatment ◽  
Differentiation Therapy ◽  
Cd11b Expression ◽  
Nb4 Cells ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Nbt Reduction ◽  
Therapeutic Doses

Abstract The introduction of ATRA-based differentiation therapy has significantly enhanced outcomes in patients with APL. However, retinoic acid syndrome and ATRA-resistance remain significant concerns. It would therefore be useful to develop drugs that reduce the therapeutic doses of ATRA needed, and would be effective in ATRA-resistant cases. We have shown previously that curcumin, the yellow compound isolated from spice turmeric, suppresses the initiation and promotion stages of cancer development. In the present study we evaluated whether curcumin affects differentiation of NB4 APL cells. The NB4 cells were derived from a patient with APL, and differentiate in response to ATRA, while NB4-R1 cells are resistant to ATRA. Treatment of NB4 cells with 5 μM curcumin enhanced ATRA-mediated differentiation. Differentiation was assessed by evaluating CD11b expression, nitroblue tetrazolium (NBT) reduction and by morphologic examination. This curumin-mediated enhanced differentiation was apparent at 1 μM as well as 0.1 μM of ATRA. Curcumin alone did not cause differentiation of the NB4 cells, although higher concentrations of curcumin caused apoptosis. We then examined the effect of curcumin on the ATRA-resistant NB-R1 cells. Addition of ATRA and curcumin together induced differentiation of the NB4-R1 cells, whereas either agent alone did not cause differentiation. The differentiation was characterized by increased CD11b expression, NBT reduction and the typical morphologic changes. In addition, differentiation of the NB4-R1 cells was accompanied by restoration of the PML-oncogenic domains (PODs). These results indicate that curcumin may be another unconventional therapeutic agent in APL, following the successful use of ATRA and arsenic trioxide.

Aminopeptidase Inhibitor Bestatin Potentiates the Induced-Differentiation Activity of All-trans-Retinoic Acid in NB4 Cells Possibly through Down-Regulating c-myc Expression.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4443-4443
Author(s):  
Mao-fang Lin ◽  
Xi-jun Qian
Keyword(s):  
Retinoic Acid ◽  
Tumor Therapy ◽  
Expression Level ◽  
Induced Differentiation ◽  
Cd11b Expression ◽  
Nb4 Cells ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Pharmacologic Activity ◽  
Nbt Reduction

Abstract All-trans-retinoic acid (ATRA) represents the sole example of clinically useful cyto-differentiating agent. ATRA treatment alone results in complete remission of nearly 80% patients with acute promyelocytic leukemia (APL). However, the therapeutic use of this compound is limited by a number of problems, including the systemic toxicity and ATRA resistant leukemia. One way to circumvent these problems is to identify the agents capable of enhancing the pharmacologic activity of ATRA. As we know, an aminopeptidase inhibitor, bestatin, had been used as an immunomodulator in anti-tumor therapy. Recently, we have reported bestatin can induce apoptosis in HL-60 and K562 cells. In the present study, we investigated whether bestatin can potentiate the ATRA induced-differentiation of APL cell line NB4 cells and whether changes of transcription factors expression are involved in this course. The cellular morphology observed by optical microscopy, the expression level of CD11b measured by flow cytometry and the nitroblue-tetrazolium (NBT) reduction assay was performed to determine the cyto-differentiation in NB4 cells. The mRNA expression levels of c-myc and c-EBPε in NB4 cells were detected by RT-PCR. NB4 cells incubated with 10nM ATRA plus 100μg/ml bestatin showed more morphologic character of metamyelocyte and band neutrophil than that of the cells treated by ATRA alone. Compared with 10nM ATRA used alone, after treating NB4 cells for 72 hours, the addition of various concentration of bestatin (50μg/ml, 75μg/ml, 100μg/ml) dose-dependently enhancesd NBT reduction of NB4 cells (17.6±2.5 vs. 12.0±2.2, p<0.05; 23.5±3.2 vs. 12.0±2.2, p<0.01; 36.0±8.3 vs. 12.0±2.2, p<0.01, respectively). 100μg/ml bestatin time-dependently increased 10nM ATRA induced NBT reduction of NB4 cells from 24 to 72 hours (p<0.01). The effect of various concentration of ATRA in combination with 100μg/ml bestatin was statistically different with the sum of the effects of individual drugs after subtracting the value of background (31.2±9.1 vs. 12.7±4.3, p<0.01; 39.5±5.0 vs.16.0±1.8, p<0.001; 49.6±5.3 vs. 22.1±1.6, p<0.001, respectively). Moreover, 10nM ATRA plus 100μg/ml bestatin could prominently elevate CD11b expression in NB4 cells compared with ATRA alone treated NB4 cells group(60.58±9.18% vs. 31.95±5.52%, p<0.01), while 100μg/ml bestatin could not induced significant changes in the expression level of CD11b in NB4 cells after 72 hours incubation. The various concentration (50μg/ml, 75μg/ml, 100μg/ml) of bestatin synergizes with 10nM ATRA to down-regulate the expression level of c-myc mRNA (p<0.01), which was inversely correlated with the NBT reduction activity of NB4 cells induced by 10nM ATRA plus various concentration bestatin (r=−0.917, p=0.028). However, 100μg/ml bestatin plus 10nM ATRA could not induce any significant changes in the expression level of c-EBPε mRNA compared with ATRA treated alone group. In conclusion, an aminopeptidase inhibitor bestatin can potentiate ATRA-induced differentiation of NB4 cells, which may be through down-regulating the expression of c-myc in concert with ATRA. Bestatin would be useful in anti-APL therapy by enhancing the pharmacologic activity of ATRA.

MLL-AF9 Positive Acute Myeloid Leukemia Cells Are Sensitive To All-Trans Retinoic Acid

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3827-3827
Author(s):  
Kenichi Sakamoto ◽  
Toshihiko Imamura ◽  
Mio Yano ◽  
Hideki Yoshida ◽  
Atsushi Fujiki ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Morphological Changes ◽  
Monocytic Differentiation ◽  
Cd11b Expression ◽  
Qrt Pcr ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Nbt Reduction

Abstract Background Among the subtypes of acute myeloid leukemia (AML), acute promyelocytic leukemia (APL) responds dramatically to differentiation therapy with all-trans retinoic acid (ATRA). But, ATRA is not sufficient to induce differentiation in non-APL AML. Herein, we first evaluated whether MLL fusion partners, such as MLL-AF9 and MLL-AF4/AF5q31, affect the sensitivity of ATRA in human and murine MLL fusion positive AML cell lines. In addition, we also assessed the level of H3K4me2 modification for the RARα gene in human AML cell lines, and whether the LSD1 inhibitor affected the ATRA-resistant MLLfusion positive AML cell lines. Methods Three human AML cell lines with MLL fusion (THP1 and MOLM13 expressing MLL-AF9, and KOCL48 expressing MLL-AF4) and two murine leukemic cell lines derived from murine Lin- hematopoietic progenitors transduced by retroviral vector expressing MLL fusion genes, such as MLL-AF9 and MLL-AF5q31 were used in this study. To test the sensitivity of ATRA, all cell lines were treated with 1 μM ATRA for three days. Monocytic differentiation was assessed by morphological analysis, NBT reduction test and flow cytometric analysis (FCM) of CD11b expression. Quantitative RT-PCR (qRT-PCR) analysis and western blotting was carried out to measure the RARα, C/EBPα, C/EBPε, and PU.1 expressions. Cytotoxicity assay was performed to determine the IC50 of ATRA in these cell lines and whether ATRA could decrease the IC50 of cytarabine in MLL-AF9positive cells by using WST assays. Chromatin immunoprecipitation (ChIP) assay was performed to determine the value of H3K4me2 status using RARα-specific primers. To determine whether tranylcypromine (TCP), which is a nonreversible LSD1 inhibitor, could reactivate ATRA sensitivity, we treated KOCL48 with 10 μM TCP and 1μM ATRA. Results We first determined that morphological changes characteristic of monocytic differentiation, CD11b expression and NBT reduction are more readily induced by ATRA in human and murine MLL-AF9 positive cells than MLL-AF4/AF5q31 positive cells. The NBT reduction percentage was 12.5±3.77 in THP1, 13.1±2.02 in MOLM13, but 7.00±2.64 in KOCL48 cells (p<0.05). The ATRA treatment also induced growth inhibition and increased gene expression related to monocytic differentiation through retinoic acid (RA) pathway, more efficiency in MLL-AF9 positive cells than MLL-AF4/AF5q31 cells. The IC50 of ATRA for THP1, MOLM13 and murine MLL-AF9 cells was 3.85, 1.24 and 1.95 μM, but over 10 μM for KOCL48 and murine MLL-AF5q31 cells. Furthermore, qRT-PCR and western blot revealed that ATRA increased expression level of RARα, C/EBPα, C/EBPε, and PU.1 in MLL-AF9 positive cells, but not in MLL-AF4/AF5q31 positive cells. Collectively, RA pathway is more impaired in MLL-AF4/AF5q31 positive cells than MLL-AF9 positive cells. In addition, the increase in RARα, C/EBPα, C/EBPε, and PU.1 mRNA expressions were observed in two primary MLL-AF9 positive AML cells treated with ATRA. Next, we also carried out ChIP assay and the H3K4me2/ H3 on the RARα promoter in MLL-AF9 positive cells were higher than MLL-AF4 positive cell. Furthermore, ATRA and TCP combination treatment in KOCL48 induced morphological changes, CD11b expression, and increased the expression level of RARα, C/EBPα, C/EBPε, and PU.1, suggesting that inhibition of LSD1 restores ATRA sensitivity. Finally, ATRA in combination with cytarabine treatment in MLL-AF9 positive cells enhanced cytarabine sensitivity: the IC50 of cytarabine in THP1, MOLM13, and murine MLL-AF9cells was 4.18, 0.04, and0.065 μM without ATRA and 0.13, 0.0005, and 0.015 μM with ATRA, respectively. Conclusions Our data demonstrated that RA pathway was more profoundly impaired in MLL-AF4/AF5q31 positive cells than MLL-AF9 positive cells, suggesting type of MLL fusion protein contributes inactivation of RA pathway. Our data also identified the sensitivity of ATRA was correlated with the ratio of H3K4me2/ H3 on the RARα promoter, and TCP restore the sensitivity of ATRA in KOCL48, suggesting the decrease of the H3K4me2/H3 plays a role in inactivation of RA pathway. Additionally, we revealed that synergistic antileukemic activity of ATRA in combination with cytarabine in MLL-AF9 positive cells. Therefore, ATRA in combination with cytarabine might be novel therapeutic option for the ATRA sensitive AML cells, especially for MLL-AF9 positive cells. Disclosures: No relevant conflicts of interest to declare.

Restoration of CCAAT Enhancer Binding Protein Alpha P42 Overcomes Myeloid Differentiation Block and All-Trans Retinoic Acid Resistance in Human Acute Promyelocytic Leukemia NB4-R1 Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1238-1238 ◽  
Author(s):  
Limengmeng Wang ◽  
Haowen Xiao ◽  
Xing Zhang ◽  
Weichao Liao ◽  
Shan Fu ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Acute Promyelocytic Leukemia ◽  
Morphological Changes ◽  
Promyelocytic Leukemia ◽  
Mtor Pathway ◽  
Myeloid Differentiation ◽  
Differentiation Therapy ◽  
Cd11b Expression ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid

Abstract All-trans retinoic acid (ATRA) is one of the first line agents in differentiation therapy for acute promyelocytic leukemia (APL). However, drug resistance becomes a major problem which influences the efficacy of ATRA. Mechanisms of ATRA resistance are ugly needed to be identified. Here we found that expression of C/EBPα, an important transcription factor for myeloid differentiation, was significantly suppressed in ATRA resistant APL cell line NB4-R1 compared with ATRA sensitive NB4 cells. Moreover, two forms of C/EBPα were unequally suppressed in NB4-R1 cells. Suppression of the full-length form P42 was more pronounced than the truncated form P30. Inhibition of PI3K/Akt/mTOR pathway was also observed in NB4-R1 cells. Meanwhile, C/EBPα expression was reduced by PI3K inhibitor LY294002 and mTOR inhibitor RAD001 in NB4 cell, suggesting that inactivation of the PI3K/Akt/mTOR pathway was responsible for C/EBPα suppression in APL cells. Then, we restored C/EBPα P42 and P30 by lentivirus vectors in NB4-R1 cells respectively, and found C/EBPα P42, but not P30, could increase CD11b, CD14, G-CSFR and GM-CSFR expression, which indicated the occurrence myeloid differentiation. Moreover, further upregulating of CD11b expression and differential morphological changes were found in NB4-R1 cells with restored C/EBPα P42 after ATRA treatment. However, CD11b expression and differential morphological changes could not be induced by ATRA in NB4-R1 cells infected with P30 expressing or control vector. Thus, we inferred that ATRA sensibility of NB4-R1 cells was enhanced by restoration of C/EBPα P42. In addition, we used histone deacetylase inhibitor trichostatin (TSA) to restore C/EBPα expression in NB4-R1 cells. Similar enhancement of myeloid differentiation and cell growth arrest were detected. Together, this study demonstrated that suppression of C/EBPα P42 induced by PI3K/Akt/mTOR inhibition impaired the differentiation and ATRA sensibility of APL cells. Restoring C/EBPα P42 is an attractive approach for differentiation therapy in ATRA resistant APL. Disclosures No relevant conflicts of interest to declare.

Interferon-γ Enhances PML Protein Expression in Acute Promyelocytic Leukemia Cells and Cooperates with All-Trans Retinoic Acid to Induce Maturation of NB4 and MR2 Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5040-5040
Author(s):  
Pengcheng He ◽  
Mei Zhang ◽  
Xiaoning Wang ◽  
Jun Qi ◽  
Jieying Xi ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Cell Line ◽  
Interferon Γ ◽  
Promyelocytic Leukemia ◽  
Nb4 Cells ◽  
Inhibition Effect ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Ifn Γ ◽  
Nbt Reduction

Abstract More than 90% patients with untreated acute promyelocytic leukemia(APL) obtain complete remission clinically by using All-trans retinoic acid(ATRA), a strong differentiation inducer. However, the rapid development of ATRA-resistance brings a new problem to the treatment of APL. Interferon(IFN), as an important cytokine, has broad biological activities. It can not only inhibit the growth of tumor cells, but also reverse the drug-resistance of chemotherapy. As proved by some research, the mechanisms of ATRA-resistance are probably related to lacking some important proteins which synthesized by Interferon-γ (IFN-γ). In order to explore a new way to solve the problem of ATRA-resistance in APL, we investigate the effect and mechanisms of IFN-γ in combination with ATRA on the proliferation/differentiation of NB4 cells(APL cell line with ATRA-sensitiveness) and MR2 cells(APL cell line with ATRA-resistance) respectively. ATRA, IFN-γ and IFN-γ in combination with ATRA were incubated with NB4 and MR2 cells respectively. The cell proliferation was tested by MTT assay, the cell differentiation was tested through light microscope, by NBT reduction test and flow cytometry(FCM). The results showed that ATRA could inhibit the growth of NB4 cells significantly (P&lt;0.05), but it had no effect on the growth of MR2 cells (P&gt;0.05). IFN-γ could inhibit the growth of both NB4 cells and MR2 cells slightly (P&lt;0.05). Moreover, the growth inhibition effect of IFN-γ in combination with ATRA on NB4 and MR2 cells was obviously stronger than that of any single drug group (P&lt;0.05). The results of cell morphology observation, NBT reduction test and CD11b antigen detection showed that ATRA could induce differentiation of NB4 cells significantly (P&lt;0.05), but it had no effect on MR2 cells (P&gt;0.05). Although IFN-γ alone had no effect on the differentiation of either NB4 or MR2 cells (P&gt;0.05), it could augment the differentiation of NB4 cells induced by ATRA (P&lt;0.05) and induce the differentiation of MR2 cells slightly (P&lt;0.05) when it combined with ATRA. Furthermore, we have observed the expression of promyelocytic leukemia(PML) protein by indirect immune fluorescent test. The results showed that the number of fluorescent particles in both NB4 and MR2 cells’ nucleus was increased significantly (P&lt;0.05) when they were incubated with IFN-γ respectively, which indicated IFN-γ could induce the expression of PML protein, a tumor growth inhibitor. It can be seen that IFN-γ could augment the proliferation inhibition effect of ATRA on NB4 and MR2 cells through enhancing the expression of PML protein. Moreover, IFN-γ in combination with ATRA not only can strengthen the induction differentiation effect of ATRA on NB4 cells, but also can partially induce the maturation of MR2 cells with ATRA-resistance.

scholarly journals DNA Methylation Predicts the Response of Triple-Negative Breast Cancers to All-Trans Retinoic Acid

Cancers ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 397 ◽  
Author(s):  
Krysta Coyle ◽  
Cheryl Dean ◽  
Margaret Thomas ◽  
Dejan Vidovic ◽  
Carman Giacomantonio ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Retinoic Acid ◽  
Cell Lines ◽  
Triple Negative ◽  
Atra Treatment ◽  
Response Elements ◽  
Acid Binding Protein ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid

All-trans retinoic acid (atRA) regulates gene expression and is used to treat acute promyelocytic leukemia. Attempts to use atRA in breast cancer without a stratification strategy have resulted in limited overall effectiveness. To identify biomarkers for the treatment of triple-negative breast cancer (TNBC) with atRA, we characterized the effects of atRA on the tumor growth of 13 TNBC cell lines. This resulted in a range of effects that was not predictable based on previously hypothesized predictors of response, such as the levels of atRA nuclear shuttling proteins fatty acid binding protein 5 (FABP5) and cellular retinoic acid binding protein 2 (CRABP2). Transcriptional profiling revealed that atRA induced distinct gene expression changes in the sensitive versus resistant cell lines that were mostly independent of the presence of retinoic acid response elements (RAREs) or peroxisome proliferator response elements (PPREs). Given the importance of DNA methylation in regulating gene expression, we hypothesized that differential DNA methylation could predict the response of TNBCs to atRA. We identified over 1400 sites that were differentially methylated between atRA resistant and sensitive cell lines. These CpG sites predicted the response of four TNBC patient-derived xenografts to atRA, and we utilized these xenografts to refine the profile and identified that as many as 17% of TNBC patients could benefit from atRA treatment. These data illustrate that differential methylation of specific CpGs may be useful biomarkers for predicting the response of patient tumors to atRA treatment.

scholarly journals Regulation of myeloblastin messenger RNA expression in myeloid leukemia cells treated with all-trans retinoic acid

Blood ◽  
1993 ◽  
Vol 81 (2) ◽  
pp. 475-481 ◽  
Author(s):  
C Labbaye ◽  
J Zhang ◽  
JL Casanova ◽  
M Lanotte ◽  
J Teng ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Mrna Expression ◽  
Messenger Rna ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Nb4 Cells ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid

Abstract Retinoic acid is known to induce differentiation of human myeloid leukemia cells in vitro. Recently, all-trans retinoic acid has been used to induce remissions in patients with acute promyelocytic leukemia, probably through differentiation of the leukemia cells. Myeloblastin (mbn) is a protease that has been identified in the human leukemia cell line HL-60. Downregulation of this protease can inhibit proliferation and induce differentiation of HL-60-derived leukemia cells. Here we have investigated the regulation of mbn messenger RNA (mRNA) expression in two human leukemia cell lines, HL-60 and NB4, treated with all-trans retinoic acid. Under this treatment, downregulation of mbn mRNA was observed in both cell lines, but was considerably delayed in NB4 cells that carry the t(15;17) translocation characteristic of acute promyelocytic leukemia. We have found that multiple mechanisms were involved in the control of mbn mRNA expression. These mechanisms were different in HL-60 and NB4 cells. Our results show that in HL-60 cells, all-trans retinoic acid rapidly decreased transcription of mbn. In contrast, in the t(15;17)-positive NB4 cells treated with all-trans retinoic acid, upregulation of mbn mRNA expression was followed by a late downregulation, both achieved via posttranscriptional mechanisms.

scholarly journals All-Trans Retinoic Acid Enhances Bacterial Flagellin-Stimulated Proinflammatory Responses in Human Monocyte THP-1 Cells by Upregulating CD14

2019 ◽  
Vol 2019 ◽  
pp. 1-11
Author(s):  
Thi Xoan Hoang ◽  
Jong Hyeok Jung ◽  
Jae Young Kim
Keyword(s):  
Retinoic Acid ◽  
Cell Surface ◽  
Human Monocyte ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Dependent Manner ◽  
Atra Treatment ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Proinflammatory Responses

All-trans retinoic acid (ATRA), an active form of vitamin A, exerts immunomodulatory functions. In this study, we examined the immune potentiating effect of ATRA on bacterial flagellin-induced NF-κB activation and proinflammatory cytokine production in human monocytic cell line THP-1. ATRA treatment significantly enhanced the flagellin-induced NF-κB/AP-1 activity in THP-1 via the RAR/RXR pathway. Similarly, ATRA enhanced the expression and production of TNF-α and IL-1β in THP-1 cells upon flagellin challenge. The cell surface expression of toll-like receptor 5 (TLR5), which is the receptor for bacterial flagellin, was significantly reduced by ATRA in a concentration- and time-dependent manner. To determine the mechanisms underlying the ATRA-enhanced immune response against bacterial flagellin despite the reduced cell surface expression of TLR5 in ATRA-treated THP-1, we examined the cell surface expression of CD14, which has been proposed to be a TLR co-receptor that enhances the response to microbial components. The cell surface expression of CD14 was significantly enhanced by ATRA treatment, especially in the presence of flagellin. Anti-CD14 antibody treatment prior to ATRA and flagellin treatments completely abolished ATRA-enhanced TNF-α and IL-1β production. Our results suggest that ATRA enhances flagellin-stimulated proinflammatory responses in human monocyte THP-1 cells by upregulating CD14 in a RAR/RXR-dependent manner.

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