RESUMO
Pharmacological induction of fetal hemoglobin has proven to be a promising therapeutic intervention in ß-hemoglobinopathies by reducing the globin chain imbalance and inhibiting sickle cell polymerization. Fagonia indica has shown therapeutic relevance to ß-thalassemia. Therefore, we study the ethnopharmacological potential of Fagonia indica and its biomarker compounds for their HbF induction ability for the treatment of ß-thalassemia. Here, we identify, compound 8 (triterpenoid glycosides) of F. indica. as a prominent HbF inducer in-vitro and in-vivo. Compound 8 showed potent erythroid differentiation, enhanced cellular proliferation, ample accumulation of total hemoglobin, and a strong notion of γ-globin gene expression in K562 cultures. Compound 8 treatment also revealed strong induction of erythroid differentiation and fetal hemoglobin mRNA and protein in adult erythroid precursor cells. This induction was associated with simultaneous downregulation of BCL11A and SOX6, and overexpression of the GATA-1 gene, suggesting a compound 8-mediated partial mechanism involved in the reactivation of fetal-like globin genes. The in vivo study with compound 8 (10 mg/kg) in ß-YAC mice resulted in significant HbF synthesis demonstrated by the enhanced level of F-cells (84.14 %) and an 8.85-fold increase in the γ-globin gene. Overall, the study identifies compound 8 as a new HbF-inducing entity and provides an early "proof-of-concept" to enable the initiation of preclinical and clinical studies in the development of this HbF-inducing agent for ß-thalassemia.
Assuntos
Hemoglobinopatias , Triterpenos , Talassemia beta , Humanos , Animais , Camundongos , gama-Globinas/genética , gama-Globinas/metabolismo , Talassemia beta/tratamento farmacológico , Talassemia beta/genética , Talassemia beta/metabolismo , Glicosídeos/farmacologia , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , Células K562 , Fatores de Transcrição , Expressão Gênica , Proteínas RepressorasRESUMO
Hypoxia and acidosis are ubiquitous hallmarks of the tumor microenvironment (TME), and in most solid cancers they have been linked to rewired cancer cell metabolism. These TME stresses are linked to changes in histone post-translational modifications (PTMs) such as methylation and acetylation, which lead to tumorigenesis and drug resistance. Hypoxic and acidotic TME cause changes in histone PTMs by impacting the activities of histone-modifying enzymes. These alterations are yet to be extensively explored in oral squamous cell carcinoma (OSCC), one of the most prevalent cancers in developing countries. Hypoxic, acidotic, and hypoxia with acidotic TME affecting histone acetylation and methylation in the CAL27 OSCC cell line was studied using LC-MS-based proteomics. The study identified several well-known histone marks, in the context of their functionality in gene regulation, such as H2AK9Ac, H3K36me3, and H4K16Ac. The results provide insights into the histone acetylation and methylation associated with hypoxic and acidotic TME, causing changes in their level in a position-dependent manner in the OSCC cell line. Hypoxia and acidosis, separately and in combination, cause differential impacts on histone methylation and acetylation in OSCC. The work will help uncover tumor cell adaptation to these stress stimuli in connection with histone crosstalk events.
Assuntos
Acidose , Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Humanos , Histonas/genética , Histonas/metabolismo , Metilação , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço , Acetilação , Microambiente Tumoral , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Processamento de Proteína Pós-TraducionalRESUMO
Exosomes are nanoscale extracellular vesicles which regulate intercellular communication. They have great potential for application in nanomedicine. However, techniques for their isolation are limited by requirements for advanced instruments and costly reagents. In this study, we developed a lyophilization-based method for isolating exosomes from cultured cells. The isolated exosomes were characterized for protein content using Bradford assay, and for size distribution and shape using scanning electron microscopy (SEM) and nanoparticles tracking analysis (NTA). In addition, CD63, CD9, CD81, HSP70 and TSG101 were evaluated as essential exosomal surface markers using Western blot. Drug loading and release studies were performed to confirm their drug delivery properties using an in vitro model. Exosomes were also loaded with commercial dyes (Cy5, Eosin) for the evaluation of their drug delivery properties. All these characterizations confirmed successful exosome isolation with measurements of less than 150 nm, having a typical shape, and by expressing the known exosome surface protein markers. Finally, tyrosine kinase inhibitors (dasatinib and ponatinib) were loaded on the exosomes to evaluate their anticancer effects on leukemia cells (K562 and engineered Ba/F3-BCR-ABL) using MTT and Annexin-PI assays. The expression of MUC1 protein on the exosomes isolated from MCF-7 cells also indicated that their potential diagnostic properties were intact. In conclusion, we developed a new method for exosome isolation from cultured cells. These exosomes met all the essential requirements in terms of characterization, drug loading and release ability, and inhibition of proliferation and apoptosis induction in Ph+ leukemia cells. Based on these results, we are confident in presenting the lyophilization-based exosome isolation method as an alternative to traditional techniques for exosome isolation from cultured cells.
Assuntos
Exossomos , Vesículas Extracelulares , Leucemia , Humanos , Exossomos/metabolismo , Células Cultivadas , Indicadores e Reagentes , Leucemia/metabolismoRESUMO
Fetal hemoglobin (HbF) is a potent genetic modifier, and the γ-globin gene induction has proven to be a sustainable therapeutic approach for the management of ß-thalassemia. In this study, we have evaluated the HbF induction ability of A. vasica in vitro and in vivo, and the identification of potential therapeutic compounds through a bioassay-guided approach. In vitro benzidine-Hb assay demonstrated strong erythroid differentiation of K562 cells by A. vasica extracts. Subsequently, an in vivo study with an aqueous extract of A. vasica (100 mg/kg) showed significant induction of the γ-globin gene and HbF production. While in the acute study, the hematological and biochemical indices were found to be unaltered at the lower dose of A. vasica. Following the bioassay-guided approach, two isolated compounds, vasicinol (1) and vasicine (2) strongly enhanced HbF levels and showed prominent cellular growth kinetics with ample accumulation of total hemoglobin in K562 cultures. High HbF levels were examined by immunofluorescence and flow cytometry analysis, concomitant with the overexpression in the γ-globin gene level. Compound 1 (0.1 µM) and compound 2 (1 µM) resulted in a greater increase in F-cells (90 and 83%) with marked up (8-fold and 5.1-fold) expression of the γ-globin gene, respectively. Molecular docking studies indicated strong binding affinities of (1) and (2) with HDAC2 and KDM1 protein that predict the possible mechanism of compounds in inhibition of these epigenetic regulators in the γ-globin gene reactivation. Altogether, these observations demonstrated the therapeutic usefulness of A. vasica for fostering HbF production in clinical implications for blood disorders.
RESUMO
The red blood cell membrane loses its integrity during hemoglobinopathies like ß-thalassemia and sickle cell disease. Various mutations have been associated with ß-thalassemia, the most prevalent of which is the IVS-1-5 (G > C) mutation. It is associated with poor prognosis of the disease with a dependency on transfusion. Here, we have investigated the effect of IVS mutation and the administration of hydroxyurea on the red blood cell membrane lipidome isolated from patients using a liquid chromatography coupled to tandem mass spectrometry based approach to identify changes in the red blood cell membrane lipidome of patients with/without the mutation and being/not being administered hydroxyurea. A total of 50 patients, with/without hydroxyurea treatment, were recruited and 62 lipid species were identified in all groups after statistical analyses using fold change analysis, ANOVA and lipids with higher VIP values extracted from the OPLS-DA loading plot. The presence of the IVS mutation showed altered expression levels of various lipid species as compared to non-IVS individuals, such as phosphatidylcholines, steroids, phenol lipids and fatty acids. Significant changes were though found with the administration of hydroxyurea where both the IVS and non-IVS groups showed a marked increase in complex lipids of the membrane, while a decrease was observed in those without hydroxyurea administration showing degradation of these membrane lipids. This study is the first to report changes incurred by IVS mutation and hydroxyurea administration in red blood cell membranes extracted from ß-thalassemia patients. Hydroxyurea administration has been perceived to improve the lipid profile of the red blood cell membrane in both IVS and non-IVS patients.
Assuntos
Talassemia beta , Humanos , Hidroxiureia/farmacologia , Hidroxiureia/uso terapêutico , Lipidômica , Lipídeos , Mutação , Talassemia beta/tratamento farmacológico , Talassemia beta/genéticaRESUMO
RBCs membrane loses its integrity during hemoglobinopathies such as ß-thalassemia and sickle cell disease. The severity of ß-thalassemia has been historically linked to the presence of XMN polymorphism which is believed to ameliorate the severity. Here, we investigate the effect of XMN polymorphism on RBC membrane lipidome isolated from patients, using LC-MS/MS based approach. A total of 50 patients were recruited and 28 lipid species were identified in all groups after statistical analyses using volcano plot and ANOVA-SCA, and lipids with higher VIP values extracted from OPLS-DA loading plot. Alteration in lipid levels specifically the membrane lipids such as PC and fatty acids were observed. Samples with XMN polymorphism exhibited up-regulation of lipids involved in membrane stability such as cholenoic acid while PC (O-41:1) was down-regulated when compared to non-XMN samples. Additionally, HU administration to samples also had profound effect on the lipids of patients in both groups. A trend of improvement in the membrane lipids was observed in patients with XMN polymorphism. HU administration has proven to further improve the membrane integrity by upregulating certain membrane lipids in such patients. The study presents a comprehensive analysis of RBC membrane lipidome with respect to the genetic variation and HU administration.
Assuntos
Talassemia beta , Cromatografia Líquida , Humanos , Hidroxiureia , Lipidômica , Lipídeos de Membrana , Espectrometria de Massas em Tandem , Talassemia beta/genéticaRESUMO
Pharmacologically induced production of fetal hemoglobin (HbF) is a pragmatic therapeutic strategy for the reduction of globin chain imbalance and improving the clinical severities of patients with ß-hemoglobinopathies. To identify highly desirable new therapeutic HbF-inducing agents, we screened functionally diverse ten monoterpenes, as molecular entities for their potent induction and erythroid differentiation ability in human erythroleukemia cell line (K562) and transgenic mice. Benzidine hemoglobin staining demonstrated six compounds to have significantly induced erythroid differentiation of K562 cells in a dose and time-dependent manner. This induction paralleled well with the optimal accumulated quantity of total hemoglobin in treated cultures. The cytotoxic studies revealed that three (carvacrol, 3-carene, and 1,4-cineole) of the six compounds with their maximal erythroid expansion ability did not affect cell proliferation and were found non-toxic. Four compounds were found to have high potency, with 4-8-fold induction of HbF at both transcriptional and protein levels in vitro. Subsequently, an in vivo study with the three active non-cytotoxic compounds showed significant overexpression of the γ-globin gene and HbF production. Carvacrol emerged as a lead HbF regulator suggested by the increase in expression of γ-globin mRNA content (5.762 ± 0.54-fold in K562 cells and 5.59 ± 0.20-fold increase in transgenic mice), accompanied by an increase in fetal hemoglobin (F-cells) levels (83.47% in K562 cells and 79.6% in mice model). This study implicates monoterpenes as new HbF inducing candidates but warrants mechanistic elucidation to develop them into potential therapeutic drugs in ß-thalassemia and sickle cell anemia.
Assuntos
Eritrócitos/efeitos dos fármacos , Eritropoese/efeitos dos fármacos , Hemoglobina Fetal/biossíntese , Hematínicos/farmacologia , Monoterpenos/farmacologia , gama-Globulinas/biossíntese , Anemia Falciforme/sangue , Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/genética , Animais , Cromossomos Artificiais de Levedura , Cimenos/farmacologia , Eritrócitos/metabolismo , Hemoglobina Fetal/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Células K562 , Camundongos Transgênicos , Regulação para Cima , Talassemia beta/sangue , Talassemia beta/tratamento farmacológico , Talassemia beta/genética , gama-Globulinas/genéticaRESUMO
Pharmacologically mediated reactivation of γ-globin gene with an increase in fetal hemoglobin production, is a cost effective experimental therapeutic intervention for the management of ß-hemoglobinopathies. Investigation of new pharmacological agents as HbF inducers from natural resources is desirable to develop safe and effective HbF inducers. We evaluated selected cinchona alkaloids (cinchonidine and quinidine) for their potential of erythroid differentiation and augmentation of fetal hemoglobin production. K562 cells were used as in vitro experimental model. Erythroid differentiation of K562 cells was studied using a benzidine assay, and total hemoglobin was estimated through a calorimetric method. Whereas, quantitative real-time PCR (qRT-PCR) was used to analyse γ-globin gene expression, and flow cytometry and immunofluorescence microscopy for evaluating HbF production. Cinchona alkaloids showed dose dependent erythroid differentiation, time driven cellular proliferation, with kinetics of hemoglobin accumulation in K562 cells. The findings of qRT-PCR showed an increase in expression of γ-globin mRNA content (3.17-fold in cinchonidine and 2.03-fold increase in quinidine treated K562 cells), accompanied by an increase in fetal hemoglobin production. Altogether, this study demonstrates that cinchona alkaloids can be used as therapeutic agents in treating ß-thalassemia after further biological investigation.
RESUMO
Fetal hemoglobin (HbF) induction is a cost-effective therapeutic approach for the treatment of ß-hemoglobinopathies like ß-thalassemia and sickle cell anemia. The present study discusses the potential of thiourea derivatives as new class of compounds that induce the fetal hemoglobin production. HbF inducing effect of thiourea derivatives was studied using experimental cell system, the human erythroleukemic K562â¯cell line. Erythroid induction of K562â¯cells was studied by the benzidine/H2O2 reaction, total hemoglobin production was estimated by plasma hemoglobin assay kit, and γ-globin gene expression by RT-qPCR, whereas fetal hemoglobin production was estimated by flow cytometry and immunofluorescence microscopy. The results indicated that newly synthesized thiourea derivative are potent inducers of erythroid differentiation of K562â¯cells with an increased γ-globin gene expression and fetal hemoglobin production. Moreover, these compounds showed no cytotoxic effect and inhibition on K562â¯cells at HbF inducing concentrations. It is important to note that hydroxyurea is a cytotoxic chemotherapeutic agent and have deleterious side effects, reflecting the need to identify new safe and effective HbF induces. These results signify thiourea derivatives as promising HbF inducers, with the potential to be studied against hematological disorders, including ß-thalassemia and sickle cell anemia.