STUDIES ON THE INDUCTION OF FOETAL HAEMOGLOBIN AND MOLECULAR MECHANISMS OF THE UP-REGULATION OF THE γ-GLOBIN GENE IN HUMANS USING PLANT INDUCERS OF FOETAL HAEMOGLOBIN
STUDIES ON THE INDUCTION OF FOETAL HAEMOGLOBIN AND MOLECULAR MECHANISMS OF THE UP-REGULATION OF THE γ-GLOBIN GENE IN HUMANS USING PLANT INDUCERS OF FOETAL HAEMOGLOBIN
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Date
2014
Authors
AIMOLA, Idowu Asegame
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Abstract
Five plants used in ethnomedicine to treat sickle cell anemia (SCA) were screened for their ability to induce Foetal Haemoglobin (HbF) synthesis in primary human Erythroid progenitor stem cells (hEPSCs) isolated from SCA individuals. A systematic literature search using MEDLINE, CAB Abstracts, EMBASE and Google Scholar was used to rank plants to be included in this study using the Research Initiative on Traditional Anti-Malaria Method (RITAM) scoring criteria. RITAM score revealed Telfairia occidentalis (TO) leaves, Azadiractha indica (AI) leaves, Adansonia digitata (AD) seeds, Terminalia catappa (TC) leaves and Sorghum bicolor (SB) leaves with the highest potentials for foetal hemoglobin induction and thus were selected for in vitro screening of their proliferative, differentiation, HbF induction and cytotoxicity effects on hEPSCs. The aqueous extracts of TO, AI, TC and SB induced differentiation of peripheral blood mononuclear cells (PBMCs) to erythroid Burst forming erythroid units (BFUe) and higher synthesis of foetal hemoglobin compared to the controls (erythropoietin (EPO) 1U/μl and HU 1μg/ml). Following fractionation using n-hexane, ethyl acetate, petroleum ether, acetone, ethanol and distilled water, the solvent fractions revealed varying effects on hEPSCs differentiation and HbF synthesis. TO ethyl acetate fraction and TC distilled water fraction (TCDWF) and n-hexane fractions (A and B) showed significantly (P<0.05) higher HbF induction of 37.8, 38.5, 28.5 and 20.8% respectively compared to the other plant fractions. TCDWF revealed a 6.8 and 9.2 fold HbF increase in both EPO-dependent and EPO-independent hEPSCs cultures respectively which was higher compared to other active fractions and controls. Caspase-3 activity was significantly (P˂0.05) by all the active fractions in EPO-dependent hEPSCs, while in EPO-independent hEPSCs, the active fractions appeared to significantly (P˂0.05) lower caspase-3 activity compared to EPO control. TCDWF fraction however, was differentially cytotoxic causing release of LDH in EPO-dependent hEPSCs while no LDH release was detected when EPO-independent hEPSCs were treated with the active fraction suggesting that TCDWF was apparently cytotoxic to EPO-dependent hEPSCs while being protective to EPO-independent cells. GC-MS of un-derivatized T. catappa HbF inducing fraction revealed several potential HbF inducing compounds (9-octadecenoic acid (Oleic acid), 2-hexadecenoic acid (2-HDA), Cis-vaccenic acid (CVA), Apigenin, Docosahexadecenoic acid (DHA), Eicosapentanoic acid (EPA) and Gentisic acid) which were further screened for their γ-globin and HbF inducing capacity on K562, JK-1 and primary transgenic mice bone marrow stem cell (TMbmEPSCs) cultures. Apigenin, DHA, Oleic acid and 2-HDA induced differentiation of erythroleukemic K562 cells and TMbmEPSCs. Apigenin, DHA and 2-HDA also induced γ-globin gene expression in K562 cells. Apigenin was most effective at 75μM while DHA and 2-HDA were effective at 6.5μM and 17μM respectively. Alkaline denaturation assay revealed that 2-HDA increased HbF synthesis significantly (P<0.05) higher than the un-induced controls. 2-HDA induction of K562 HbF was detected 24 hours post induction. Cis-vaccenic acid (CVA) similarly induced differentiation of K562 cells in a concentration
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dependent fashion but failed to induce γ-globin gene expression in this cell type. CVA (50μM) did not significantly (P>0.05) alter JK-1 cell survival as assessed by Trypan blue staining. Likewise, CVA at 50μM induced JK1 and TMbmEPSCs γ-globin gene expression. CVA induced γ-globin expression and increased γ-globin mRNA incrementally as assayed by qRT-PCR. CVA also induced significantly (P<0.05) increased JK-1 HbF synthesis. CVA (50μM) did not alter EKLF1 gene expression in JK-1 cells however we observed over a 100 fold increase in Bcl11a gene expression in JK-1 cells. CVA elevated TMbmEPSCs EKLF gene 8 fold relative to the un-induced cells. Erythropoietin (EPO) pre-differentiation of JK-1 cells before CVA induction did not significantly (P>0.05) alter CVA induced differentiation of JK-1 cells, although a 2 fold increase in γ-globin gene expression was observed in EPO (2U/ml) pre-differentiated cells compared to un-differentiated JK-1 cells. Paradoxically, a significant inhibition of γ-globin gene expression was observed when CVA (50μM) was added simultaneously with EPO to either EPO pre-differentiated or un-differentiated JK-1 cultures. Inhibition of CVA biosynthesis using 40μM Cycloate suppressed the γ-globin inductive effects of CVA in JK-1 and TMbmEPSCs. In addition, inhibition of fatty acid elongase V (Elovl5) using 10μM Isoxyl also suppressed the γ-globin inductive effects CVA in JK-1 and transgenic mice bone marrow progenitor stem cells. CVA (50μM) failed to completely rescue the inhibitory effects of Cycloate and Isoxyl 48 hours post inhibition in JK-1 cells, suggesting that CVA induced γ-globin expression is mediated through a downstream biosynthetic product of CVA. Isoxyl inhibition significantly (P<0.05) enhanced CVA induced BFUe potential of TMbmEPSCs while Cycloate inhibition significantly (P<0.05) reduced that potential. The results suggest that CVA directly induce hEPSC differentiation and indirectly modulate β-globin gene expression in humans through mechanisms which involve increased transcription and translation of the γ-globin gene, with minimal cytotoxic effects on human Our findings provide important clues for further evaluations of CVA as a potential HbF therapeutic inducer and a clinical substitute of HU for SCA management
Description
A DISSERTATION SUBMITTED TO THE SCHOOL OF POST GRADUATE STUDIES, AHMADU BELLO UNIVERSITY, ZARIA IN PARTIAL FULFILMENT FOR THE AWARD OF PhD BIOCHEMISTRY Department of Biochemistry, Ahmadu Bello University, Zaria Nigeria.
Keywords
STUDIES,, INDUCTION,, FOETAL HAEMOGLOBIN,, MOLECULAR MECHANISMS,, UP-REGULATION,, γ-GLOBIN GENE,, HUMANS,, PLANT INDUCERS,, FOETAL HAEMOGLOBIN,