Design, Synthesis and Biological Evaluation of A Boron Containing Retinoid As a Novel Therapeutic Agent for Acute Promyelocytic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5008-5008
Author(s):  
Carolina D. Schinke ◽  
Bhaskar C Das ◽  
Swati Goel ◽  
Tushar Bhagat ◽  
Sangeeta Nischal ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Retinoic Acid ◽  
Cell Line ◽  
Acute Promyelocytic Leukemia ◽  
Cell Lines ◽  
Therapeutic Agent ◽  
Fold Increase ◽  
Resistant Cell ◽  
Biological Evaluation ◽  
Promyelocytic Leukemia

Abstract Abstract 5008 Cure rates of acute leukemia remain poor emphasizing the need to for novel therapies. All trans retinoic acid (ATRA) is an effective therapeutic agent in a subtype of acute myeloid leukemia and relieves transcriptional repression induced by the PML-RAR oncoprotein by binding to the retinoic acid receptor. Even though ATRA is effective, the treatment course is characterized by a high rate of toxicity and ATRA resistance is seen in some cases of acute promyelocytic leukemia. In an attempt to improve outcomes, we devised a methodology for creation of boronic acid and other newer retinoic acid analogues. Our lead compound, MA-21 was generated by replacing the terminal carboxyl group of ATRA with a boronic ester using Wittig reactions. Computation modeling revealed that MA-21 can fit in the RARa pocket and can form increased covalent bonding with cysteine residues within the receptor. As opposed to other synthetic retinoids, the addition of a boron atom resulted in significantly enhanced cytotoxicity in leukemic cell lines, even those that were resistant to ATRA. MA-21 at 1uM dose led to significant reduction in proliferation of ATRA sensitive NB4 APL cell line (1.8 fold decrease after 96hrs, p= 0.028) as well as in ATRA resistant cell lines NB4.007/6 (3.3 fold decrease after 72hrs and 2.1 decrease after 96hrs, p values of 0.018 and 0.046) and NB4.306 (2.6 fold decrease after 96hrs, p= 0.032). MA-21 was able to induce these effects by inducing significant G2/M cell cycle arrest and not by increased apoptosis or cellular differentiation. Cell cycle was assessed by Flow Cytometry after 96hrs of incubation and showed a significant increase in G2/M percentage in the ATRA sensitive and resistant cell lines compared to DMSO (NB4 cell line- 1.35 fold increase, p= 0.035; NB4.007/6- 1.35 fold increase, p= 0.015 and NB4.306- 2 fold increase, p= 0.023). Thus, we demonstrate novel synthetic methodology to synthesize boron containing novel retinoids and demonstrate the potential of these compounds as therapeutic agents in resistant leukemias.Figure1:Structures of ATRA and MA-21Figure1:. Structures of ATRA and MA-21 Disclosures: No relevant conflicts of interest to declare.

Novel Retinoic Acid Metabolism Blocking Agents (RAMBAs) Induce Differentiation in ATRA Resistant Cell Line: Potential New Theraputics for Acute Promyleocytic Leukemia (APL).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 746-746
Author(s):  
Kavita B. Kalra ◽  
Xiangfei Cheng ◽  
Marion Womak ◽  
Christopher Gocke ◽  
Jyoti B. Patel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Retinoic Acid ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Lines ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Breast Cancer Cell Lines ◽  
Hematopoietic Stem

Abstract All trans retinoic acid (ATRA) has been used in differentiation therapy for APL and other types of cancers. However, the rapid emergence of ATRA resistance due in part to ATRA-induced acceleration of ATRA metabolism limits its use. A novel strategy to overcome the limitation associated with exogenous ATRA therapy has been developed by inhibiting the cytochrome P450-dependent ATRA-4-hydroxylase enzyme responsible for ATRA metabolism. These inhibitors are referred to as RAMBAs. Novel RAMBAs were developed which demonstrated a superior apoptosis, cell growth inhibition, in vivo anti-tumor effect in addition to the differentiation effect in breast cancer cell lines (Patel JB et al. J. Med. Chem2004,47:6716). We tested 3 RAMBAs, VN/14-1, 50-1, and 66-1 to investigate their activities against APL cell lines. RAMBAs did not confer cytotoxicity or apoptosis induction in vitro at the concentration between 0.5 to 5 μM as opposed to breast or prostate cancer cell lines. However, the differentiation effect was demonstrated by morphological and phenotypic changes using Wright-Giemsa stain and CD11b staining measured by flow cytometric analysis. VN/14-1 and VN/66-1 induced differentiation and apoptosis morphologically and phenotypically in HL60 cells. VN/14-1 and VN/50-1 showed superior differentiation in NB4 cell line compared to ATRA (70%, 69%, and 45%, respectively). Interestingly, HL60 ATRA resistant cell line was induced to undergo differentiation by VN/14-1 (0.5μM) at 55% whereas ATRA (0.5, 1, 5μM) showed less than 5% by flow cytometry analysis. VN/14-1 inhibited cell cycle at S phase whereas ATRA did not attenuate the cell cycle at the same concentration. We also tested the effect of RAMBAs on human CD34+ enriched cell colony formation. RAMBAs were added to the methylcellulose culture plates with CD34+ cells and colonies were determined after 14 days. There was no difference in the CFU-GM or BFU-E colony count between the control and the RAMBAs group. In summary, RAMBAs are promising differentiation agents in the treatment of APL, possibly through an inhibition of Cyp26A leading to increased endogenous ATRA levels. In addition, cell cycle inhibition may be a mechanism of differentiation induction in ATRA resistant cell lines. RAMBAs did not affect normal hematopoietic stem cells. We are currently testing whether RAMBAs can induce acetylation of histones in APL cell lines.

scholarly journals Arsenic trioxide resistance in acute promyelocytic leukemia: More to it than PML mutations

2020 ◽  
Author(s):  
Nithya Balasundaram ◽  
Saravanan Ganesan ◽  
Ezhilarasi Chendamarai ◽  
Hamenth Kumar Palani ◽  
Arvind Venkatraman ◽  
...  
Keyword(s):  
Cell Line ◽  
Arsenic Trioxide ◽  
Acute Promyelocytic Leukemia ◽  
Cell Lines ◽  
Resistant Cell ◽  
Chemotherapeutic Agents ◽  
Resistant Cell Line ◽  
Promyelocytic Leukemia ◽  
Genetic Mutations ◽  
List Type

AbstractAcquired genetic mutations can confer resistance to arsenic trioxide (ATO) in the treatment of acute promyelocytic leukemia (APL). However, such resistance-conferring mutations are rare and do not explain the majority of disease recurrence seen in the clinic. We have generated a stable ATO resistant promyelocytic cell from a ATO sensitive NB4 cell line. We also noted that another ATRA resistant cell line (UF1) was cross resistant to ATO. We have characterized these resistant cell lines and observed that they significantly differed in their immunophenotype, drug transporter expression, drug resistance mutation profile and were also cross-resistant to other conventional chemotherapeutic agents. The NB4 derived resistant cell line had the classical A216V PML-B2 domain mutation while the UF1 cell line did not. Gene expression profiling revealed prominent dysregulation of the cellular metabolic pathways in the resistant cell lines. Glycolytic inhibition by 2-DG was efficient and comparable to the standard of care (ATO) in targeting the sensitive APL cell lines and was also effective in the in vivo transplantable APL mouse model; however, it did not affect the ATO resistant cell lines. The survival of the resistant cell lines was significantly affected by compounds targeting the mitochondrial respiration irrespective of the existence of ATO resistance-conferring genetic mutations. Our data demonstrate the addition of mitocans can overcome ATO resistance. We further demonstrated that the combination of ATO and mitocans has the potential in the treatment of non-M3 AML and the translation of this approach in the clinic needs to be explored further.Key pointsMetabolic rewiring promotes ATO resistance, which can be overcome by targeting mitochondrial oxidative phosphorylation.Combination of ATO and mitocans can be exploited as a potential therapeutic option for relapsed APL and in non-M3 AML patients.

scholarly journals Metabolic adaptation drives arsenic trioxide resistance in acute promyelocytic leukemia

2021 ◽  
Author(s):  
Nithya Balasundaram ◽  
Saravanan Ganesan ◽  
Ezhilarasi Chendamarai ◽  
Hamenth Kumar Palani ◽  
Arvind Venkatraman ◽  
...  
Keyword(s):  
Cell Line ◽  
Arsenic Trioxide ◽  
Acute Promyelocytic Leukemia ◽  
Cell Lines ◽  
Resistant Cell ◽  
Promyelocytic Leukemia ◽  
Metabolic Adaptation ◽  
Genetic Mutations ◽  
Apoptotic Protein

Acquired genetic mutations can confer resistance to arsenic trioxide (ATO) in the treatment of acute promyelocytic leukemia (APL). However, such resistance-conferring mutations are rare and do not explain most disease recurrence seen in the clinic. We have generated stable ATO-resistant promyelocytic cell lines that are also less sensitive to ATRA and the combination of ATO and ATRA compared to the sensitive cell line. Characterization of these in-house generated resistant cell lines showed significant differences in immunophenotype, drug transporter expression, anti-apoptotic protein dependence, and PML-RARA mutation. Gene expression profiling revealed prominent dysregulation of the cellular metabolic pathways in these ATO resistant APL cell lines. Glycolytic inhibition by 2-DG was sufficient and comparable to the standard of care (ATO) in targeting the sensitive APL cell line. 2-DG was also effective in the in vivo transplantable APL mouse model; however, it did not affect the ATO resistant cell lines. In contrast, the resistant cell lines were significantly affected by compounds targeting the mitochondrial respiration when combined with ATO, irrespective of the ATO resistance-conferring genetic mutations or the pattern of their anti-apoptotic protein dependency. Our data demonstrate that the addition of mitocans in combination with ATO can overcome ATO resistance. We further show that this combination has the potential in the treatment of non-M3 AML and relapsed APL. The translation of this approach in the clinic needs to be explored further.

Novel Anthraquinone Derivatives as Dual Inhibitors of Topoisomerase 2 and Casein Kinase 2: In Silico Studies, Synthesis and Biological Evaluation on Leukemic Cell Lines

2019 ◽  
Vol 18 (11) ◽  
pp. 1551-1562 ◽  
Author(s):  
Abbas Kabir ◽  
Kalpana Tilekar ◽  
Neha Upadhyay ◽  
C.S. Ramaa
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Lines ◽  
Biological Evaluation ◽  
Casein Kinase ◽  
Casein Kinase 2 ◽  
Cell Cycle Analysis ◽  
Protein Targets ◽  
Topoisomerase 2 ◽  
Dual Inhibitors

Background: Cancer being a complex disease, single targeting agents remain unsuccessful. This calls for “multiple targeting”, wherein a single drug is so designed that it will modulate the activity of multiple protein targets. Topoisomerase 2 (Top2) helps in removing DNA tangles and super-coiling during cellular replication, Casein Kinase 2 (CK2) is involved in the phosphorylation of a multitude of protein targets. Thus, in the present work, we have tried to develop dual inhibitors of Top2 and CK2. Objective: With this view, in the present work, 2 human proteins, Top2 and CK2 have been targeted to achieve the anti-proliferative effects. Methods: Novel 1-acetylamidoanthraquinone (3a-3y) derivatives were designed, synthesized and their structures were elucidated by analytical and spectral characterization techniques (FTIR, 1H NMR, 13C NMR and Mass Spectroscopy). The synthesized compounds were then subjected to evaluation of cytotoxic potential by the Sulforhodamine B (SRB) protein assay, using HL60 and K562 cell lines. Ten compounds were analyzed for Top2, CK2 enzyme inhibitory potential. Further, top three compounds were subjected to cell cycle analysis. Results: The compounds 3a to 3c, 3e, 3f, 3i to 3p, 3t and 3x showed excellent cytotoxic activity to HL-60 cell line indicating their high anti-proliferative potential in AML. The compounds 3a to 3c, 3e, 3f, 3i to 3p and 3y have shown good to moderate activity on K-562 cell line. Compounds 3e, 3f, 3i, 3x and 3y were found more cytotoxic than standard doxorubicin. In cell cycle analysis, the cells (79-85%) were found to arrest in the G0/G1 phase. Conclusion: We have successfully designed, synthesized, purified and structurally characterized 1- acetylamidoanthraquinone derivatives. Even though our compounds need design optimization to further increase enzyme inhibition, their overall anti-proliferative effects were found to be encouraging.

Chromosome-mediated gene transfer of hydroxyurea resistance and amplification of ribonucleotide reductase activity

1983 ◽  
Vol 3 (6) ◽  
pp. 1053-1061
Author(s):  
W H Lewis ◽  
P R Srinivasan
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Ribonucleotide Reductase ◽  
Chinese Hamster ◽  
Fold Increase ◽  
Resistant Cell ◽  
Plating Efficiency ◽  
Wild Type Cell ◽  
Wild Type ◽  
Metaphase Chromosomes

Metaphase chromosomes purified from a hydroxyurea-resistant Chinese hamster cell line were able to transform recipient wild-type cells to hydroxyurea resistance at a frequency of 10(-6). Approximately 60% of the resulting transformant clones gradually lost hydroxyurea resistance when cultivated for prolonged periods in the absence of drug. One transformant was subjected to serial selection in higher concentrations of hydroxyurea. The five cell lines generated exhibited increasing relative plating efficiency in the presence of the drug and a corresponding elevation in their cellular content of ribonucleotide reductase. The most resistant cell line had a 163-fold increase in relative plating efficiency and a 120-fold increase in enzyme activity when compared with the wild-type cell line. The highly hydroxyurea-resistant cell lines had strong electron paramagnetic resonance signals characteristic of an elevated level of the free radical present in the M2 subunit of ribonucleotide reductase. Two-dimensional electrophoresis of cell-free extracts from one of the resistant cell lines indicated that a 53,000-dalton protein was present in greatly elevated quantities when compared with the wild-type cell line. These data suggest that the hydroxyurea-resistant cell lines may contain an amplification of the gene for the M2 subunit of ribonucleotide reductase.

AM580, a stable benzoic derivative of retinoic acid, has powerful and selective cyto-differentiating effects on acute promyelocytic leukemia cells

Blood ◽  
1996 ◽  
Vol 87 (4) ◽  
pp. 1520-1531 ◽  
Author(s):  
M Gianni ◽  
M Li Calzi ◽  
M Terao ◽  
G Guiso ◽  
S Caccia ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Cell Line ◽  
Acute Promyelocytic Leukemia ◽  
Retinoic Acid Receptor ◽  
Chromosomal Translocation ◽  
Promyelocytic Leukemia ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
Stable Phase ◽  
Differentiation Markers

All-trans retinoic acid (ATRA) is successfully used in the cyto- differentiating treatment of acute promyelocytic leukemia (APL). Paradoxically, APL cells express PML-RAR, an aberrant form of the retinoic acid receptor type alpha (RAR alpha) derived from the leukemia- specific t(15;17) chromosomal translocation. We show here that AM580, a stable retinobenzoic derivative originally synthesized as a RAR alpha agonist, is a powerful inducer of granulocytic maturation in NB4, an APL-derived cell line, and in freshly isolated APL blasts. After treatment of APL cells with AM580 either alone or in combination with granulocyte colony-stimulating factor (G-CSF), the compound induces granulocytic maturation, as assessed by determination of the levels of leukocyte alkaline phosphatase, CD11b, CD33, and G-CSF receptor mRNA, at concentrations that are 10- to 100-fold lower than those of ATRA necessary to produce similar effects. By contrast, AM580 is not effective as ATRA in modulating the expression of these differentiation markers in the HL-60 cell line and in freshly isolated granulocytes obtained from the peripheral blood of chronic myelogenous leukemia patients during the stable phase of the disease. In NB4 cells, two other synthetic nonselective RAR ligands are capable of inducing LAP as much as AM580, whereas RAR beta- or RAR gamma-specific ligands are totally ineffective. These results show that AM580 is more powerful than ATRA in modulating the expression of differentiation antigens only in cells in which PML-RAR is present. Binding experiments, using COS-7 cells transiently transfected with PML-RAR and the normal RAR alpha, show that AM580 has a lower affinity than ATRA for both receptors. However, in the presence of PML-RAR, the synthetic retinoid is a much better transactivator of retinoic acid-responsive element-containing promoters than the natural retinoid, whereas, in the presence of RAR alpha, AM580 and ATRA have similar activity. This may explain the strong cyto-differentiating potential of AM580 in PML-RAR-containing leukemic cells.

scholarly journals The PML/RAR alpha oncoprotein is a direct molecular target of retinoic acid in acute promyelocytic leukemia cells

Blood ◽  
1996 ◽  
Vol 88 (8) ◽  
pp. 2826-2832 ◽  
Author(s):  
JV Raelson ◽  
C Nervi ◽  
A Rosenauer ◽  
L Benedetti ◽  
Y Monczak ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Fusion Protein ◽  
Cell Line ◽  
Acute Promyelocytic Leukemia ◽  
Promyelocytic Leukemia ◽  
Dominant Negative ◽  
Myeloid Differentiation ◽  
Wild Type ◽  
Retinoid Receptor ◽  
Western Blots

Acute promyelocytic leukemia (APL) is characterized by the translocation, t(15;17) and the expression of a PML/RAR alpha fusion protein that is diagnostic of the disease. There is evidence that PML/RAR alpha protein acts as a dominant negative inhibitor of normal retinoid receptor function and myeloid differentiation. We now show that the PML/RAR alpha fusion product is directly downregulated in response to retinoic acid (tRA) treatment in the human APL cell line, NB4. tRA treatment induces loss of PML/RAR alpha at the protein level but not at the level of mRNA, as determined by Northern blots, by Western blots, and by ligand binding assays and in binding to RA-responsive DNA elements. We present evidence that this regulation is posttranslational. This evidence suggests that tRA induces synthesis of a protein that selectively degrades PML/RAR alpha. We further show that this loss of PML/ RAR-alpha is not limited to the unique APL cell line. NB4, because PML/RAR alpha protein is selectively downregulated by tRA when expressed in the transfected myeloid cell line U937. The loss of PML/RAR alpha may be directly linked to tRA-induced differentiation, because in a retinoid-resistant subclone of NB4, tRA does not decrease PML/RAR alpha protein expression. In NB4 cells, the specific downregulation of the fusion protein decreases the ratio of PML/RAR alpha to wild-type RAR alpha. Because the ratio of expression of PML/RAR alpha to wild-type RAR alpha and PML may be important in maintaining the dominant negative block of myelocytic differentiation, these data suggest a molecular mechanism for restoration by tRA normal myeloid differentiation in APL cells.

scholarly journals A Retinoid-Resistant Acute Promyelocytic Leukemia Subclone Expresses a Dominant Negative PML-RARα Mutation

Blood ◽  
1997 ◽  
Vol 89 (12) ◽  
pp. 4282-4289 ◽  
Author(s):  
Wenlin Shao ◽  
Laura Benedetti ◽  
William W. Lamph ◽  
Clara Nervi ◽  
Wilson H. Miller
Keyword(s):  
Gene Expression ◽  
Retinoic Acid ◽  
Ligand Binding ◽  
Cell Line ◽  
Acute Promyelocytic Leukemia ◽  
Regulation Of Gene Expression ◽  
Promyelocytic Leukemia ◽  
Dominant Negative

Abstract The unique t(15; 17) of acute promyelocytic leukemia (APL) fuses the PML gene with the retinoic acid receptor α (RARα) gene. Although retinoic acid (RA) inhibits cell growth and induces differentiation in human APL cells, resistance to RA develops both in vitro and in patients. We have developed RA-resistant subclones of the human APL cell line, NB4, whose nuclear extracts display altered RA binding. In the RA-resistant subclone, R4, we find an absence of ligand binding of PML-RARα associated with a point mutation changing a leucine to proline in the ligand-binding domain of the fusion PML-RARα protein. In contrast to mutations in RARα found in retinoid-resistant HL60 cells, in this NB4 subclone, the coexpressed RARα remains wild-type. In vitro expression of a cloned PML-RARα with the observed mutation in R4 confirms that this amino acid change causes the loss of ligand binding, but the mutant PML-RARα protein retains the ability to heterodimerize with RXRα and thus to bind to retinoid response elements (RAREs). This leads to a dominant negative block of transcription from RAREs that is dose-dependent and not relieved by RA. An unrearranged RARα engineered with this mutation also lost ligand binding and inhibited transcription in a dominant negative manner. We then found that the mutant PML-RARα selectively alters regulation of gene expression in the R4 cell line. R4 cells have lost retinoid-regulation of RXRα and RARβ and the RA-induced loss of PML-RARα protein seen in NB4 cells, but retain retinoid-induction of CD18 and CD38. Thus, the R4 cell line provides data supporting the presence of an RARα-mediated pathway that is independent from gene expression induced or repressed by PML-RARα. The high level of retinoid resistance in vitro and in vivo of cells from some relapsed APL patients suggests similar molecular changes may occur clinically.

Halofuginone Exerts Antiproliferative and Antiangiogenic Actions on Acute Promyelocytic Leukemia Cells through Modulation of the TGFβ Pathway.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2850-2850
Author(s):  
Lorena L. Figueiredo-Pontes ◽  
Ana Silvia G. Lima ◽  
Barbara A. Santana-Lemos ◽  
Ana Paula A. Lange ◽  
Luciana C. Oliveira ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Growth Inhibition ◽  
Acute Promyelocytic Leukemia ◽  
Cell Lines ◽  
Cell Cultures ◽  
Promyelocytic Leukemia ◽  
Cell Growth Inhibition ◽  
Tgfβ Signaling ◽  
Tgfβ Pathway

Abstract The effects of TGFβ signaling in tumorigenesis is both cell type and context-dependent. Although this cytokine may behave as tumor suppressor in early stages of malignant transformation, tumor progression is often accompanied by altered TGFβ responsiveness and increased angiogenesis. Acute Promyelocytic Leukemia (APL) is a distinct subtype of Acute Myelogenous Leukemia characterized by rearrangements involving the PML and RARα genes on chromosomes 15 and 17, respectively. The expression of the PML/RARα oncoprotein leads to PML delocalization and functional impairment. Among its physiological roles, PML is a regulator of the TGFβ pathway, and the expression of PML-RARα has been associated with TGFβ resistance to differentiation and cell growth inhibition. Moreover, TGFβ is known to regulate Vascular Endothelial Growth Factor (VEGF) production and response. APL patients present increased bone marrow microvessel density, and the APL cell line NB4 was shown to secrete high levels of VEGF. Our aim was to test on APL the effect of Halofuginone (HF), an alkaloid that has been shown to inhibit TGFβ in other cell types. Cell cultures of NB4 and NB4-R2 cell lines, this latter resistant to ATRA, were treated with increasing doses of HF (6.25, 12.5, 25, 50, 100 ng/ml) and 10−6M of ATRA during 72 hours. Cell proliferation and apoptosis were accessed by flow cytometry using a simultaneous staining with bromodeoxyuridine and 7AAD. In NB4, there was significant cell growth inhibition with HF doses superior to 25 ng/ml (P <0.001). In addition, a 1.5 fold increase in apoptosis was seen with 100 ng/ml (P <0.001). In NB4-R2, cell growth inhibition was observed with 50 and 100 ng/ml and apoptosis with 100 ng/ml of HF (P < 0.001). HF was able to block the cell cycle progression at G1/S transition and, simultaneously, reduce Bcl2 protein expression in both cell lines. Concomitantly, mRNA expression of TGFβ target genes involved in cell cycle regulation was evaluated by real time PCR. Results showed the upregulation of p15, SMAD3, TGFβ and TGFβRI, and downregulation of c-MYC by treatment with high doses of HF (75 and 100 ng/ml). VEFG and TGFβ production was measured by ELISA in supernatants after 72 hours of culture. Significant reduction of VEGF levels was detected in samples treated with HF at doses higher than 25 ng/ml or with ATRA (P=0.018) and a decrease of TGFβ secretion was observed with 50 and 100 ng/ml of HF (P=0.026). Nuclear extracts from cell cultures treated as above were obtained, and western blot analysis showed that higher doses of HF (50 to 100 ng/ml) reduced TGFβ and Smad 4 expression. Our results indicate that HF was able to inhibit TGFβ at protein level and consequently to reduce VEGF production and thus may revert APL aberrant angiogenesis. As TGFβ transcription is at least in part auto-regulated, HF treatment was associated with an increase of TGFβ transcripts. These effects were independent of ATRA sensitivity, since both cell lines presented the same behavior. Although the disruption of TGFβ signaling itself is not sufficient to initiate malignant transformation, it may be a critical second step that contributes to leukemia progression. In this context, HF may have therapeutic potential in APL.

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