Cristina Zuccato

Fetal Hemoglobin Inducers from the Natural World: A Novel Approach for Identification of Drugs for the Treatment of β-Thalassemia and Sickle-Cell Anemia

The objective of this review is to present examples of lead compounds identified from biological material (fungi, plant extracts and agro-industry material) and of possible interest in the field of a pharmacological approach to the therapy of β-thalassemia using molecules able to stimulate production of fetal hemoglobin (HbF) in adults. Concerning the employment of HbF inducers as potential drugs for pharmacological treatment of β-thalassemia, the following conclusions can be reached: (i) this therapeutic approach is reasonable, on the basis of the clinical parameters exhibited by hereditary persistence of fetal hemoglobin patients, (ii) clinical trials (even if still limited) employing HbF inducers were effective in ameliorating the symptoms of β-thalassemia patients, (iii) good correlation of in vivo and in vitro results of HbF synthesis and γ-globin mRNA accumulation indicates that in vitro testing might be predictive of in vivo responses and (iv) combined use of different inducers might be useful to maximize HbF, both in vitro and in vivo. In this review, we present three examples of HbF inducers from the natural world: (i) angelicin and linear psoralens, contained in plant extracts from Angelica arcangelica and Aegle marmelos, (ii) resveratrol, a polyphenol found in grapes and several plant extracts and (iii) rapamycin, isolated from Streptomyces hygroscopicus.

Effects of rapamycin on accumulation of α-, β- and γ-globin mRNAs in erythroid precursor cells from β-thalassaemia patients

Abstract:  We studied the effects of rapamycin on cultures of erythroid progenitors derived from the peripheral blood of 10 β‐thalassaemia patients differing widely with respect to their potential to produce foetal haemoglobin (HbF). For this, we employed the two‐phase liquid culture procedure for growing erythroid progenitors, high performance liquid chromatography for analysis of HbF production and reverse transcription polymerase chain reaction for quantification of the accumulation of globin mRNAs. The results demonstrated that rapamycin induced an increase of HbF in cultures from all the β‐thalassaemia patients studied and an increase of their overall Hb content/cell. The inducing effect of rapamycin was restricted to γ‐globin mRNA accumulation, being only minor for β‐globin and none for α‐globin mRNAs. The ability of rapamycin to preferentially increase γ‐globin mRNA content and production of HbF in erythroid precursor cells from β‐thalassaemia patients is of great importance as this agent (also known as sirolimus or rapamune) is already in clinical use as an anti‐rejection agent following kidney transplantation. These data suggest that rapamycin warrants further evaluation as a potential therapeutic drug in β‐thalassaemia and sickle cell anaemia.

Involvement of miRNA in erythroid differentiation

miRNAs are a family of small ncRNAs that regulate gene expression by targeting mRNAs in a sequence-specific manner, inducing translational repression or mRNA degradation. In this review, we present and discuss the available literature on the expression of miRNAs in erythroid cells. There are several experimental systems that can be employed for studies focusing on the relationship between miRNAs and erythroid differentiation, including human embryonic stem cells forced to erythroid differentiation, K562 and UT-7 cells induced to hemoglobin production by chemical compounds, erythropoietin-treated erythroid precursor cells from normal subjects or patients affected by hematological disease and in vivo systems, such as zebrafish embryos. Several miRNAs were identified as deeply involved in the erythroid phenotype, including miR-15a, miR-16-1, miR-126, miR-144, miR-451 and miR-210. Several functions related with erythroid cells were demonstrated to be regulated by these miRNAs, including maturation and proliferation of early erythroid cells, expression of fetal γ-globin genes and enucleation. These identified erythroid specific miRNAs represent the starting point to develop new protocols for miRNA therapeutics, based on both anti-miR molecules or miRNA replacement.

Everolimus Is a Potent Inducer of Erythroid Differentiation and γ-Globin Gene Expression in Human Erythroid Cells

We studied the effects of everolimus on the erythroid differentiation of human leukaemic K562 cells and on the cultures of erythroid progenitors derived from the peripheral blood of β-thalassaemia patients. A quantitative real-time reverse-transcription polymerase chain reaction assay was employed for the quantification of the accumulation of globin mRNAs. The results obtained demonstrate that everolimus is a potent inducer of the erythroid differentiation of K562 cells. Erythroid induction is associated with an increase in α- and γ-globin mRNAs. In erythroid precursor cells from 4 β-thalassaemia patients, everolimus stimulated a preferential increase (ranging from 1.8- to 7.2-fold) in γ-globin mRNA. Only minor effects were observed on the expression of α-globin genes. These results, in our opinion, are of interest as this compound is already employed in clinical trials as an anti-rejection agent following kidney transplantation. These data suggest that everolimus warrants further evaluation as a potential therapeutic drug in the treatment of β-thalassaemia.

Resveratrol: Antioxidant activity and induction of fetal hemoglobin in erythroid cells from normal donors and β-thalassemia patients

Thalassemia and sickle-cell anemia (SCA) present a major public health problem in countries where the number of carriers and affected individuals is high. As a result of the abnormalities in hemoglobin production, cells of thalassemia and SCA patients exhibit oxidative stress, which ultimately is responsible for the chronic anemia observed. Therefore, identification of compounds exhibiting both antioxidant and hemoglobin-inducing activities is highly needed. Our results demonstrate resveratrol to be such a compound. This was shown both in the human K562 cell line, as well as in erythroid precursors derived from normal donors and β-thalassemia patients. Resveratrol was shown to exhibit antioxidant activity and to stimulate the expression of the γ-globin genes and the accumulation of fetal hemoglobin (HbF). To the best of our knowledge, this is the first report pointing to such a double effect of resveratrol. Since this natural product is already marketed as an antioxidant, future investigations should concentrate on demonstrating its potential to augment HbF production in experimental animal models (e.g., thalassemia and SCA mice) as well as in patients. We believe that the potential of clinical use of resveratrol as an antioxidant and HbF stimulator may offer a simple and inexpensive treatment to patients.

Recent trends in the gene therapy of β-thalassemia.

The β-thalassemias are a group of hereditary hematological diseases caused by over 300 mutations of the adult β-globin gene. Together with sickle cell anemia, thalassemia syndromes are among the most impactful diseases in developing countries, in which the lack of genetic counseling and prenatal diagnosis have contributed to the maintenance of a very high frequency of these genetic diseases in the population. Gene therapy for β-thalassemia has recently seen steadily accelerating progress and has reached a crossroads in its development. Presently, data from past and ongoing clinical trials guide the design of further clinical and preclinical studies based on gene augmentation, while fundamental insights into globin switching and new technology developments have inspired the investigation of novel gene-therapy approaches. Moreover, human erythropoietic stem cells from β-thalassemia patients have been the cellular targets of choice to date whereas future gene-therapy studies might increasingly draw on induced pluripotent stem cells. Herein, we summarize the most significant developments in β-thalassemia gene therapy over the last decade, with a strong emphasis on the most recent findings, for β-thalassemia model systems; for β-, γ-, and anti-sickling β-globin gene addition and combinatorial approaches including the latest results of clinical trials; and for novel approaches, such as transgene-mediated activation of γ-globin and genome editing using designer nucleases.

Bergamot (Citrus bergamia Risso) Fruit Extracts as γ-Globin Gene Expression Inducers: Phytochemical and Functional Perspectives

Epicarps of Citrus bergamia fruits from organic farming were extracted with the objective of obtaining derived products differently rich in coumarins and psoralens. The extracts were chemically characterized by (1)H nuclear magnetic resonance (NMR), gas chromatography-flame ionization detection (GC-FID), gas chromatography-mass spectrometry (GC-MS), and high-pressure liquid chromatography (HPLC) for detecting and quantifying the main constituents. Both bergamot extracts and chemical standards corresponding to the main constituents detected were then assayed for their capacity to increase erythroid differentiation of K562 cells and expression of γ-globin genes in human erythroid precursor cells. Three experimental cell systems were employed: (a) the human leukemic K562 cell line, (b) K562 cell clones stably transfected with a pCCL construct carrying green-enhanced green fluorescence protein (EGFP) under the γ-globin gene promoter, and (c) the two-phase liquid culture of human erythroid progenitors isolated from healthy donors. The results suggest that citropten and bergapten are powerful inducers of differentiation and γ-globin gene expression in human erythroid cells. These data could have practical relevance, because pharmacologically mediated regulation of human γ-globin gene expression, with the consequent induction of fetal hemoglobin, is considered to be a potential therapeutic approach in hematological disorders, including β-thalassemia and sickle cell anemia.

Production of β-globin and adult hemoglobin following G418 treatment of erythroid precursor cells from homozygous β039 thalassemia patients

In several types of thalassemia (including β039‐thalassemia), stop codon mutations lead to premature translation termination and to mRNA destabilization through nonsense‐mediated decay. Drugs (for instance aminoglycosides) can be designed to suppress premature termination, inducing a ribosomal readthrough. These findings have introduced new hopes for the development of a pharmacologic approach to the cure of this disease. However, the effects of aminoglycosides on globin mRNA carrying β‐thalassemia stop mutations have not yet been investigated. In this study, we have used a lentiviral construct containing the β039‐thalassemia globin gene under control of the β‐globin promoter and a LCR cassette. We demonstrated by fluorescence‐activated cell sorting (FACS) analysis the production of β‐globin by K562 cell clones expressing the β039‐thalassemia globin gene and treated with G418. More importantly, after FACS and high‐performance liquid chromatography (HPLC) analyses, erythroid precursor cells from β039‐thalassemia patients were demonstrated to be able to produce β‐globin and adult hemoglobin after treatment with G418. This study strongly suggests that ribosomal readthrough should be considered a strategy for developing experimental strategies for the treatment of β0‐thalassemia caused by stop codon mutations. Am. J. Hematol., 2009.

Increase in γ-globin mRNA content in human erythroid cells treated with angelicin analogs

The aim of the present study was to identify molecular analogs of angelicin (ANG) able to increase erythroid differentiation of K562 cells and expression of γ-globin genes in human erythroid precursor cells, with low effects on apoptosis. ANG-like molecules are well-known photosensitizers largely used for their antiproliferative activity in the treatment of different skin diseases (i.e., psoriasis, vitiligo, eczema, and mycosis fungoides). To verify the activity of these derivatives, we employed three experimental cell systems: (1) the human leukemic K562 cell line, (2) K562 cell clones stably transfected with a pCCL construct carrying green-EGFP under the γ-globin gene promoter, and (3) the two-phase liquid culture of human erythroid progenitors isolated from normal donors and β-thalassemia patients. The results of our study suggest that trimethyl ANG is a powerful inducer of erythroid differentiation, compared with known inducers, such as ANG, cytosine arabinoside, mithramycin, and cisplatin. These data could have practical relevance, because pharmacologically mediated regulation of human γ-globin gene expression, with the consequent induction of fetal hemoglobin, is considered a potential therapeutic approach in hematological disorders including β-thalassemia and sickle cell anemia.

Increase of microRNA-210, Decrease of Raptor Gene Expression and Alteration of Mammalian Target of Rapamycin Regulated Proteins following Mithramycin Treatment of Human Erythroid Cells

Expression and regulation of microRNAs is an emerging issue in erythroid differentiation and globin gene expression in hemoglobin disorders. In the first part of this study microarray analysis was performed both in mithramycin-induced K562 cells and erythroid precursors from healthy subjects or β-thalassemia patients producing low or high levels of fetal hemoglobin. We demonstrated that: (a) microRNA-210 expression is higher in erythroid precursors from β-thalassemia patients with high production of fetal hemoglobin; (b) microRNA-210 increases as a consequence of mithramycin treatment of K562 cells and human erythroid progenitors both from healthy and β-thalassemia subjects; (c) this increase is associated with erythroid induction and elevated expression of γ-globin genes; (d) an anti-microRNA against microRNA-210 interferes with the mithramycin-induced changes of gene expression. In the second part of the study we have obtained convergent evidences suggesting raptor mRNA as a putative target of microRNA-210. Indeed, microRNA-210 binding sites of its 3’-UTR region were involved in expression and are targets of microRNA-210-mediated modulation in a luciferase reporter assays. Furthermore, (i) raptor mRNA and protein are down-regulated upon mithramycin-induction both in K562 cells and erythroid progenitors from healthy and β-thalassemia subjects. In addition, (ii) administration of anti-microRNA-210 to K562 cells decreased endogenous microRNA-210 and increased raptor mRNA and protein expression. Finally, (iii) treatment of K562 cells with premicroRNA-210 led to a decrease of raptor mRNA and protein. In conclusion, microRNA-210 and raptor are involved in mithramycin-mediated erythroid differentiation of K562 cells and participate to the fine-tuning and control of γ-globin gene expression in erythroid precursor cells.