Quantitative Analysis of γ-Globin Gene Expression in Human Erythroleukaemic K562 Cells Treated with Theobroma cacao Bark Ethanolic Extract

Therapeutic reactivation of foetal haemoglobin (HbF) can alleviate clinical complications associated with sickle cell anaemia (SCA), a severe monogenic disorder that is prevalent in sub-Saharan Africa. Traditional medicine practitioners in Nigeria utilize the stem bark of Theobroma cacao as a haematinic for SCA patients, but its potential to induce HbF remains uncertain. Here, we investigated the cytotoxic effects of ethanol extract of Theobroma cacao bark (TCBEE) using the MTT assay and quantified in real-time, γ-globin gene expression levels in human erythroleukaemic K562 cells treated with TCBEE (1, 5, 10 and 30 µg/mL) or 200 µM hydroxyurea (HU) for 24 hours, alongside untreated cells. Total RNA (A260/A280 > 1.9) isolated from un/treated cells was converted to cDNA for RT-qPCR analysis, and the gene expression data was normalized with GAPDH as a reference gene. TCBEE demonstrated moderate cytotoxicity against K562 cells, which are of leukaemic origin, with an IC50 value of 101.5 µg/mL in the MTT assay, suggesting that TCBEE might have good anti-cancer components. The relative quantification of the γ-globin gene revealed a decrease in fold changes as the concentration of TCBEE increased. Notably, treatment with 1 µg/mL TCBEE showed a significant (p < 0.0001) 1.08-fold change compared to HU-treated cells which could be beneficial for individuals with haemoglobinopathies, such as SCA. Therefore, this study pinpoints the therapeutic potential of TCBEE in SCA management and recommends further research to identify the bioactive compounds in TCBEE responsible for naturally inducing γ-globin gene expression.