Claude Dupont1 Eric Couillerot2 Reynald Gillet1 Catherine Caron3 Monique Zeches-Hanrot3 Jean-FrancËois Riou1 Chantal Trentesaux1
The Benzophenanthridine Alkaloid Fagaronine Induces Erythroleukemic Cell Differentiation by Gene Activation
Fagaronine, a benzophenanthridine alkaloid from Fagara zanthoxyloides Lam. (Rutaceae), has been tested on the erythroleukemic cell line K562 in order to explain some previous results on cell differentiation. In this study we showed that fagaronine induces a significant hemoglobinization of the human erythroleukemic cell line K562. This hemoglobin synthesis was accompanied by a strong increase of erythroid mRNA expression such as g- and a-globin, and PBGD, an enzyme of heme synthesis. In addition, the Epo-R transcripts were also stimulated indicating that cells are engaged in a maturation process. Both transcription factors GATA-1 and NF-E2, which play an important role in the regulation of genes involved in the erythroid differentiation, were also transcriptionally up-regulated. To elucidate the possible role of GATA-1 in the FAG-induced differentiation of K562 cells, we transfected reporter constructs containing regulatory regions of erythroid genes encompassing GATA-1 binding sites. After 48 hours of treatment, FAG stimulated the EPO-R and g-globin promoters by 2- to 3-fold and the promoter/enhancer region
Introduction Leukemic cells can be considered as maturation-arrested cells that continue to proliferate and rapidly accumulate, escaping
of GATA-1 gene by 3.2-fold. A mutation within the GATA-1 binding sites strongly decreased the promoter activation induced by FAG. Taken together, our results represent a demonstration that FAG exerts its differentiating activity by a specific activation of the regulating GATA-1 regions of genes involved in the erythroid phenotype expression.
Original Paper
Abstract
Key words Fagaronine ´ K562 cell differentiation ´ leukemia ´ erythroid gene expression ´ GATA-1 transcription factor Abbreviations ACLA: aclacinomycin FAG: fagaronine PBGD: porphobilinogene deaminase EPO-R: erythropoietin receptor GAPDH: glyceraldehydes 3-phosphate dehydrogenase FCS: fetal calf serum
the normal regulatory pathways controlling cell proliferation and differentiation. Numerous physiological as well as nonphysiological agents, including antitumor drugs, have been described to induce differentiation of leukemic cells [1]. A complete
Affiliation JE 2428 Onco-Pharmacologie, UFR Pharmacie, IFR 53 BiomolØcules, UniversitØ de Reims Champagne-Ardenne, Reims, France 2 Laboratoire de Stress, DØfenses et Reproduction des Plantes, URVVC EA2069, UFR Sciences, UniversitØ de Reims Champagne-Ardenne, Reims, France 3 Laboratoire de Pharmacognosie, FRE 2715 CNRS UFR Pharmacie, UniversitØ de Reims Champagne-Ardenne, Reims, France 1
Correspondence Chantal Trentesaux ´ JE 2428 Onco-Pharmacologie ´ UFR Pharmacie ´ IFR 53 BiomolØcules ´ UniversitØ de Reims Champagne-Ardenne ´ 51 rue Cognacq Jay ´ 51096 Reims cedex ´ France ´ Phone: +33-326-918-045 ´ Fax: +33-326-918926 ´ E-mail:
[email protected] Received August 9, 2004 ´ Accepted January 6, 2005 Bibliography Planta Med 2005; 71: 489±494 ´ Georg Thieme Verlag KG Stuttgart ´ New York DOI 10.1055/s-2005-864147 ISSN 0032-0943
489
remission by differentiation therapy was obtained in patients with acute promyelocytic leukemia treated by all-trans-retinoic acid (ATRA) [2].
Original Paper 490
Our group previously demonstrated that anthracycline antitumor drugs such as aclacinomycin (ACLA) and doxorubicin (DOX), at subtoxic concentrations, induced in vitro the erythroid differentiation of human leukemic K562 cells, leading to the appearance of hemoglobinized cells. ACLA stimulated the transcription of genes involved in hemoglobin synthesis, by the recruitment of erythroid-specific transcription factors, notably GATA-1 and NF-E2 [3], [4], [5]. In contrast, DOX induced the hemoglobinization of these cells by a post-transcriptional mechanism leading to an increased stability of the erythroid transcripts [5]. Among the other compounds able to stimulate erythroid differentiation, the benzo[c]phenanthridine alkaloid fagaronine (FAG) (Fig.1) was also reported to induce the hemoglobinization of K562 cells [6] but through an unknown mechanism. Other works have established that FAG displays an antileukemic activity against murine leukemia P388 in vivo [7], inhibits DNA polymerase activity in murine embryos [8], nucleic acid and protein synthesis in KB cells, respectively [9]. These biological activities are related to its properties to intercalate DNA and to interact with the ribosomal system [10]. FAG also inhibits the activities of the DNA topoisomerases I and II [11], [12], human DNA ligase I [13] and reverse transcriptases from RNA virus [14], especially the human HIV-1 reverse transcriptase in vitro [15]. Since ACLA was also reported to intercalate DNA and to inhibit topoisomerase I activity [16], we wondered whether the differentiating activity of FAG is triggered by similar molecular events related to transcriptional mechanisms. We have studied the erythroid gene expression induced by FAG in the human erythroid K562 cell line using RT-PCR and reporter gene analysis. Our results clearly indicate that FAG stimulates erythroid gene transcription through a mechanism involving the GATA-1 transcription factors.
Material and Methods Plant material The roots of Fagara zanthoxyloides Lam. were collected in 1999, in the Ivory Coast and identified by Dr. C. Moretti. A voucher specimen (No. 15 042) is kept at the Herbarium of the National Center of Floristics, University of Cocody, Abidjan, Ivory Coast. Extraction and isolation Dried roots of Fagara zanthoxyloides (50 g), defatted with light petroleum (1 L), were extracted with MeOH (1 L) at room tem-
Fig. 1 Structure of fagaronine.
perature. The MeOH solution, on evaporation under reduced pressure gave an extract (2.2 g) which was dissolved in 0.02 N HCl (50 mL). The aqueous solution was precipitated by Mayer's reagent (20 mL) and the precipitate (390 mg) was dissolved in MeOH-Me2CO-H2O (6 : 2 : 1). The alkaloids were converted to the chlorides by passage through an Amberlite IRA 400 (60 mL) column. After concentration under reduced pressure, a residue (212 mg) was obtained. This gave pure fagaronine (25 mg) as bright yellow needles after three crystallizations from a mixture of ethyl acetate-methanol: m. p. 202 8C followed by solidification and melting again at 255 8C; spectral data (UV, 1H-NMR 500 MHz and MS) were as given in [7]. Cell line and induction of erythroid differentiation The human erythroleukemic K562 cells were cultured in RPMI 1640 Glutamax medium, 10 % FCS (Invitrogen) as previously described [3]. FAG chloride was reconstituted in 70 % ethanol as a 0.1 M stock solution and diluted in the culture medium immediately before use. Various FAG concentrations were added to the K562 cell suspensions at the beginning of the exponential growth phase. Cell cultures were incubated in a 5 % CO2 humidified atmosphere at 37 8C during 72 hours. Growth inhibition and cell viability were evaluated as previously described [3]. After 3 days of treatment, the number of erythroid differentiated cells was determined by scoring benzidine-positive cells. K562 cells were stained using a benzidine-H2O2 method and gave an intense blue cytoplasmic staining known to correlate with hemoglobin synthesis. As previously described [6], an average of 300 cells was scored for benzidine-positivity and the results are expressed in percent. RNA extraction and RT-PCR analysis Total RNA was extracted from 5 106 cells using the TriZOL reagent (Invitrogen) as recommended by the manufacturer. One microgram of total RNA was reverse transcribed in a 20 mL reaction volume using oligo-dT primers, Superscript II reverse transcriptase (Invitrogen) according to the manufacturer's instructions. At the end of the reaction, the volume of the RT products was adjusted to 200 mL with DNase RNase-free water. A 10 mL aliquot of cDNA was used for PCR amplification using a -[32P]-dCTP (NEN) and gene specific primers of: g-globin (forward: 5¢GGCAACCTGTCCTCTGCCTC-3¢; reverse: 5¢-GCCAGGAAGCTTGCACCTCA-3¢) [17]; a-globin (forward: 5¢-TGGGGTAAGGTCGGCGCGCA-3¢; reverse: 5¢-TGCACCGCAGGGGTGAACTC-3¢) [18]; PBGD (forward: 5¢-GGTCCTACTATCGCCTCCCTC-3¢; reverse: 5¢-AGAATCTTGTCCCCTGTGGTG-3¢); Epo-R (forward: 5¢-AGCCTGTGTCGCTGCTGACGC-3¢; reverse: 5¢-GGTCCTCCGTGAAGGGGGTGC-3¢); GATA-1 (forward: 5¢-GATCCTGCTCTGGTGTCCTCC-3¢; reverse: 5¢ACAGTTGAGCAATGGGTACAC-3¢) [17]; NF-E2 (forward: 5¢-ATTTGAGCCCCAAGCCCCAGC-3¢; reverse: 5¢-CCAGCCTCTGTCCCCTCCAGC-3¢). Amplification of GAPDH was performed as control using the same PCR conditions with primer (forward: 5¢-CTCTGCCC CCTCTGCTGATGC-3¢; reverse: 5¢-CCATCACGCCACAGTTTCCCG-3¢). PCR was performed using Taq DNA polymerase (Invitrogen) with the following cycling conditions: 94 8C for 2 min, followed by 15 ± 25 cycles of 94 8C for 30 sec, 60 8C for 30 sec, and 72 8C for 60 sec and at the last cycle the reaction was maintained at 72 8C for 10 min to finish cDNA chain elongation. Amplified products were analyzed on 6 % non-denaturating polyacrylamide gels in 1 TBE. After electrophoresis, the gels were
Dupont C et al. The Benzophenanthridine Alkaloid ¼ Planta Med 2005; 71: 489 ± 494
exposed and quantified on a GS-363 Molecular Imager (BioRad).
Transient transfection of K562 cells and analysis of reporter gene signal Reporter constructs were prepared by insertion of promoter and enhancer regions in restriction sites of pGL2-basic plasmids, respectively, upstream and downstream of the firefly luciferase gene as described previously [4]. K562 cells were transfected by these reporter plasmids using the transferrinfection technique (Transferrinfection kit, Serva). Briefly, 6 mg plasmid DNA were mixed with 10 mg Fe-loaded transferrin-polylysine complex in a 0.5 mL 200 mM HEPES buffer (pH 7.2). This mix was added to pretreated (24 h incubation in culture medium containing 50 mM desferroxamine) K562 cells in a proportion of 1 mL of culture medium containing 50 mM desferroxamine and 100 mM chloroquine for 3 105 cells. After 6 h at 37 8C, for DNA capture, cells were washed once with RPMI, divided into equal parts, and then cultivated in the same medium with or without FAG for 24 or 48 h. Cells were resuspended in 0.25 mM Tris-HCl (pH 7.5) and cellular extracts were obtained by three cycles of freezing-thawing. The amount of protein in the extracts was determined by using the Bio-Rad protein assay. Luciferase activity in the extracts was tested with the Luciferase Assay Kit (Promega) in accordance with the manufacturer's instructions and quantified using a Lumac-3M luminometer (Bertold). An absolute signal was determined as the maximal rate of the sample luminescence during the first minute of the assay and the activities were finally expressed as light units/mg of protein.
Results Hemoglobinized cell content and growth inhibition of human leukemic K562 cells were evaluated after 3 days of treatment by various FAG concentrations (Fig. 2). FAG induced a dose-dependent hemoglobinization of K562 cells. The maximal differentiating effect of around 60 % was obtained at 6 mM FAG and maintained at 10 mM, as compared to untreated cells (1 % benzidinepositive cells). This induction of hemoglobin synthesis was accompanied by a marked growth inhibition varying from 63 % for the lowest concentration to 95 % for the highest. In contrast, these concentrations of FAG had a limited effect on cell viability, as determined by trypan blue dye exclusion, and cell death did not exceed 5 % at the optimal differentiating concentration (6 mM) versus 2 % in control cells.
Original Paper
GATA-1 protein analysis Western blot analysis of GATA-1 protein expression was performed as previously described [19]. Proteins were separated on a 10 % gel by SDS-PAGE and blotted onto PVDF membrane (Amersham). The membrane was blocked by a 2-h incubation in Trisbuffered saline containing 5 % non-fat milk, 0.05 % Tween 20. Immunodetections were performed by incubating the membrane with the specific GATA-1 monoclonal antibody (Santa Cruz) and then with the secondary antisera, conjugated with horse radish peroxidase (HRP). Filters were developed using the ECL Western blotting detection reagent (Amersham).
Fig. 2 Effects of FAG treatment on cell growth and differentiation of the K562 cell line. K562 cells were incubated during 72 h in the presence of various concentrations of FAG (1 ± 10 mM). The percentages of cell growth inhibition, hemoglobinization and cell viability were determined as described in Materials and Methods. Data are the means S.D (standard deviation) of three independent experiments.
The expression of erythroid mRNAs was studied after 3 days of treatment by 6 mM FAG (Fig. 3). The PCR products analysis showed that FAG induced the over-expression of g- and a- globin transcripts (2-fold), of porphobilinogene deaminase (PBGD) mRNAs (4.5-fold), a key enzyme of the heme synthesis, and of Epo-R mRNAs (2.2-fold), a receptor for the erythropoietin hormone which regulates the erythroid differentiation process (Fig. 3). Such an increased mRNA expression was found to be specific to erythroid genes since the expression of GAPDH ubiquitous transcripts remained constant after FAG treatment. Taking into account the role of GATA-1 and NF-E2 transcription factors in the regulation of erythroid gene expression, we also examined their expression following FAG treatment (Fig. 3). Similarly, FAG induced the over-expression of GATA-1 (2.7-fold) and NF-E2 transcripts (2.5-fold) after 3 days of treatment, as compared to control cells. These results suggested that FAG may exert its differentiating effects by a transcriptional regulation of erythroid gene expression. To support these data, we examined whether the over-expression of GATA-1 transcripts was also associated with an increased GATA-1 protein level. After 3 days of treatment with 6 mM FAG, cell lysates were analyzed by Western blot using a monoclonal anti GATA-1 antibody. The results shown in Fig. 4 indicate that FAG induced a 2- to 3-fold over-expression of GATA-1 protein as compared to untreated cells, in agreement with the over-expression of GATA-1 transcripts. In order to determine whether the accumulation of erythroid mRNAs resulted from a transcriptional activation mediated by GATA-1, we have transfected K562 cells with different plasmid constructs containing either the promoter of EPO-R or g-globin genes upstream to the firefly luciferase gene. These 2 promoter regions contained GATA-1 consensus sequences. Then, we analyzed the effect of FAG treatment (6 mM) for 24 or 48 hours onto the reporter activity.
Dupont C et al. The Benzophenanthridine Alkaloid ¼ Planta Med 2005; 71: 489 ± 494
491
Fig. 4 FAG increased GATA-1 protein expression. K562 cells were treated with 6 mM FAG during 3 days. Western blot analysis of GATA-1 protein was performed as described in Materials and Methods using a GATA-1 monoclonal antibody. Lane 1, K562 cells; lane 2, K562 cells + FAG. The amount of protein loaded was normalized by performing an immunoblot analysis using an anti-actin Mab. Results from one experiment are representative of three.
Original Paper
To determine whether the GATA binding sites contained in these promoter sequences played a role in the FAG-mediated gene activation, we also used a construct with the GATA-1 gene promoter/enhancer region, which included two inverted canonical GATA binding sites [4]. This construct was significantly activated after 48 hours of FAG treatment (3.2-fold activation, Fig. 5B). In parallel, a construct containing mutated GATA-1 binding sites located in the enhancer region was also used. In that case, FAG induced transcription activation by 1.9-fold only. These results indicated that the GATA-1 binding sites were involved in the promoter regulation induced by FAG treatment and thus represent a molecular basis to explain the FAG-induced transcriptional stimulation of erythroid genes.
Discussion
492
Fig. 3 FAG increased erythroid gene expression. K562 cells were induced by 6 mM FAG during 3 days. RT-PCR analysis of g-globin, a-globin, PBGD, EPO-R, GATA-1, NF-E2 and GAPDH mRNAs was performed as described in Materials and Methods in the presence of a[32P]-d-CTP. After electrophoresis on an 8 % polyacrylamide gel, PCR products were detected and quantified by exposure on a Bio-Rad GS-363 Molecular Imager. (A) Lane c, PCR negative control; lane 1, K562 cells; lane 2, K562 cells + FAG. Results from one experiment representative of three. (B) Quantification results obtained in FAG-treated cells are expressed as percentages of results in control cells. Data are the means S.D. of three independent experiments. ** and ***, values were significantly different from control according to Student's t test with p < 0.01 and p < 0.001, respectively.
As shown in Fig. 5A, luciferase activity for the g-globin construct was found to be increased in FAG-treated cells and reached a maximum at 48 hours with a 3.3-fold activation, as compared to untreated cells. Under the same conditions, the EPO-R promoter activation was also found activated by 2.0-fold after 24 hours of FAG treatment and was maintained at this level after 48 hours, (2.1-fold, Fig. 5A). As a control, the signal obtained from the pGL2-basic plasmid never exceeded 0.1 % of the activities measured for erythroid constructs (Fig. 5A). Therefore, at the optimal FAG differentiating concentration, a transcription of the reporter gene under the control of erythroid gene promoter regions was observed.
We have examined here the effects of FAG on erythroid differentiation and growth of K562 cells. FAG induced an efficient hemoglobinization of the K562 cell line without subsequent toxicity together with a strong inhibition of cell growth (about 80 %). These observations are in agreement with previous results [6] and emphasize the interest in FAG which blocks cell division through a strong growth inhibition and induction of cell differentiation. The relationship between these two effects of the drug could be explained by the fact that cell differentiation is accompanied by a loss of proliferation capacity. No lethality was observed at the concentration used, confirming the absence of an acute toxicity of fagaronine on the K562 cell line. In vitro, numerous compounds are able to induce cancer cell differentiation [1] and appear to represent an attractive alternative or adjuvant therapy to the conventional cytotoxic chemotherapy. Up to now, clinical applications of the differentiation therapy have been successfully achieved with the all-trans-retinoic acid treatment of patients with acute promyelocytic leukemia [2]. More recently, differentiation of the malignant clone and complete clinical remission has been obtained in an ATRA-refractory APL patient treated with ATRA in combination with phenyl butyrate, an HDAC inhibitor [20]. Similar results were observed in vitro on an ATRA-resistant cell line NB4 treated by a retinoid/butyric prodrug, which led to growth inhibition, partial differentiation and apoptosis of the resistant cells. This ªtranscription therapyº combines elements to facilitate transcriptional initiation of blocked differentiation pathways by inhibiting histone deacetylase [21].
Dupont C et al. The Benzophenanthridine Alkaloid ¼ Planta Med 2005; 71: 489 ± 494
and NF-E2 mRNAs as well as by GATA-1 protein accumulation. These results are in agreement with an erythroid maturation that seems to occur at the transcriptional level. Indeed, results obtained with reporter constructs containing erythroid gene regulatory regions showed that FAG caused an increased transcriptional activity of luciferase gene downstream of the g-globin, EPO-R and GATA-1 promoters. Constructs with GATA-1 gene enhancer region mutated or not at the level of two GATA-1 binding sites clearly showed that the binding of GATA-1 to its target sequence was required to stimulate reporter gene. Although we cannot exclude that other factors are involved, it is interesting to note that GATA-1's implication in FAG transcriptional activation was also described for the antitumor drug aclacinomycin [5].
Original Paper
All these data show for the first time that fagaronine exerts its differentiating activity by a specific activation of regulatory regions which control the erythroid differentiation program of human erythroleukemic cells. This process involves the participation of erythroid transcription factors such as GATA-1. Fagaronine may represent a new family of natural products able to act by modulating the activity of genes important for proliferation, differentiation and apoptosis control.
Acknowledgements This work was supported by grants from the region of Champagne-Ardenne, the Ligue Nationale contre le Cancer, ComitØs de la Haute-Marne et de l'Aisne. C.D. was the recipient of a fellowship from the Reims city. Fig. 5 Effects of FAG on the transcriptional activity of reporter constructs. (A) Activity of g-globin and EPO-R promoter (P). Luciferase activity in transfected K562 cells was determined as described in Materials and Methods after 24 or 48 hours of incubation in the presence or in the absence of FAG and expressed in lights units/mg of proteins. (B) Comparison of the luciferase activity from constructs containing the promoter and enhancer (P./E.) of the GATA-1 gene or the promoter and the mutated enhancer (P./mutated E.) of the same gene after 48 hours treatment with 6 mM FAG. Results are the means S.D. of three independent experiments. ** and ***, values significantly different from untreated control (p < 0.01 and p < 0.001, respectively) according to Student's t test. §§, activation values from mutated constructs were significantly different from those obtained in non-mutated constructs (p < 0.01, Student's t test).
In a previous work, our group was also interested in the molecular mechanisms by which antitumor compounds can induce erythroid differentiation of the human K562 leukemic cells and demonstrated that the anthracycline derivative aclacinomycin acted at the transcriptional level by stimulating GATA-1 and NF-E2, both specific transcription factors regulating the expression of erythroid genes [3], [4]. Another mechanism involving the erythroid RNA stabilization rather than its transcriptional activation was also observed for doxorubicin, another anthracycline currently used in clinical practice, suggesting that differentiating effects of antitumor agents are mediated by various molecular mechanisms. Here, we showed that hemoglobin synthesis mediated by FAG treatment was preceded by an increased transcription of several genes known as markers of erythroid differentiation (g- and aglobins, PBGD, and EPO-R) and by an over-expression of GATA-1
References 1
Reiss M, Gamba-Vitalo C, Sartorelli AC. Induction of tumor cell differentiation as a therapeutic approach: preclinical models for hematopoietic and solid neoplasms. Cancer Treat Rep 1986; 70: 201 ± 18 2 Degos L, Dombret H, Chomienne C, Daniel MT, MiclØa JM, Chastang C, Castaigne S, Fenaux P. All-trans-retinoic acid as a differentiating agent in the treatment of acute promyelocytic leukemia. Blood 1995; 85: 2643 ± 53 3 Trentesaux C, Ngo Nyoung M, Aries A, Morceau F, Ronchi A, Ottolenghi S, Jardillier JC, Jeannesson P. Increased expression of GATA-1 and NFE2 erythroid-specific transcription factors during aclacinomycin-mediated differentiation of human erythroleukemic cells. Leukemia 1993; 7: 452 ± 7 4 Aries A, Trentesaux C, Ottolenghi S, Jardillier JC, Jeannesson P, Doubeikovski A. Activation of erythroid-specific promoters during anthracycline-induced differentiation of K562 cells. Blood 1996; 87: 2885 ± 90 5 Morceau F, Chenais B, Gillet R, Jardillier JC, Jeannesson P, Trentesaux C. Transcriptional and posttranscriptional regulation of erythroid gene expression in anthracycline-induced differentiation of human erythroleukemic cells. Cell Growth Diff 1996; 7: 1023 ± 9 6 Como L, Jeannesson P, Trentesaux C, Desoize B, Jardillier JC. The antileukemic alkaloid fagaronine and the human K 562 leukemic cells: effects on growth and induction of erythroid differentiation. Leuk Res 1987; 11: 445 ± 51 7 Messmer WM, Tin-WA M, Fong HHS, Bevelle C, Farnsworth NR, Abraham DJ, Trojanek J. Fagaronine, a new tumor inhibitor isolated from Fagara zanthoxyloides Lam. (Rutaceae). J Pharm Sci 1972; 61: 1858 ± 9 8 Sethi VS. Inhibition of mammalian and oncornavirus nucleic acid polymerase activities by alkoxybenzophenantridine alkaloids. Cancer Res 1976; 36: 2390 ± 5
Dupont C et al. The Benzophenanthridine Alkaloid ¼ Planta Med 2005; 71: 489 ± 494
493
9
10
11
12
13
Original Paper
14 15
Pezzuto JM, Antosiak SK, Messmer WM, Slaytor MB, Honig GR. Interaction of the antileukemic alkaloid, 2-hydroxy-3,8,9-trimethoxy-5methylbenzo[c]phenanthridine (fagaronine), with nucleic acids. Chem Biol Interact 1983; 43: 323 ± 39 Casiano Torres CA, Baez A. Effects of the antitumor drugs 3-nitrobenzothialozo[3,2-a]quinolium and fagaronine on nucleic acid and protein synthesis. Biochem Pharmacol 1986; 35: 679 ± 85 Larsen AK, Grondard L, Couprie J, Desoize B, Como L, Jardillier JC, Riou J.F. The antileukemic alkaloid fagaronine is an inhibitor of DNA topoisomerases I and II. Biochem Pharmacol 1993; 46: 1403 ± 12 Fleury F, Sukhanova A, Ianoul A, Devy J, Kudelina I, Duval O, Alix AJ, Jardillier JC, Nabiev I. Molecular determinants of site-specific inhibition of human DNA topoisomerase I by fagaronine and ethoxidine. Relation to DNA binding. J Biol Chem 2000; 275: 3501 ± 9 Tan GT, Lee S, Lee IS, Chen J, Leitner P, Bersterman JM, Kinghorn AD, Pezzuto JM. Natural-product inhibitors of human DNA ligase I. Biochem J 1996; 314: 993 ± 1000 Sethi ML. Inhibition of reverse-transcriptase activity by benzophenantridine alkaloids. J Nat Prod 1979; 42: 187 ± 96 Tan GT, Pezzuto JM, Kinghorn AD, Hughes SH. Evaluation of natural products as inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. J Nat Prod 1991; 54: 143 ± 54
16
Sorensen BS, Jensen PB, Sehested M, Jensen PS, Kjeldsen E, Nielsen OF, Alsner J. Antagonistic effect of aclarubicin on camptothecin induced cytotoxicity: role of topoisomerase I. Biochem Pharmacol 1994; 47: 2105 ± 10 17 Leonard M, Brice M, Engel JD, Papayannopoulou T. Dynamics of GATA transcription factor expression during erythroid differentiation. Blood 1993; 82: 1071 ± 9 18 Privitera E, Schiro R, Longoni D, Ronchi A, Rambaldi A, Bernasconi S, Ottolenghi S, Masera G, Biondi A. Constitutive expression of GATA-1, EPOR, a-globin, and g-globin genes in myeloid clonogenic cells from juvenile chronic myelocytic leukemia. Blood 1995; 86: 323 ± 8 19 Gillet R, Devemy E, Dupont C, Billat C, Jeannesson P, Trentesaux C. Evidence of the role of protein kinase C during aclacinomycin induction of erythroid differentiation in K 562 cells. FEBS Lett 1999; 454: 331 ± 4 20 Warrell RPJ, He L, Richon V, Calleja E, Pandolfi PP. Therapeutic targeting of transcription in acute promyelocytic leukemia by use of an inhibitor of histone deacetylase. J Natl Cancer Inst 1998; 90: 1621 ± 5 21 Mann KK, Rephaeli A, Colosimo AL, Diaz Z, Nudelman A, Levovich I, Jing Y, Waxman S, Miller Jr WH. A retinoid/butyric acid prodrug overcomes retinoic acid resistance in leukemias by induction of apoptosis. Mol Cancer Res 2003; 1: 903 ± 12
494
Dupont C et al. The Benzophenanthridine Alkaloid ¼ Planta Med 2005; 71: 489 ± 494
