Improvement of sulforaphane production in hairy root cultures of broccoli (Brassica oleracea L. var. italica) by eliciting Myrosinase gene expression and its effect on breast cancer cells

Abstract

Plant-derived hairy root cultures have the potential to be a significant biotechnological tool for producing valuable secondary metabolite compounds. This study deals with the production of broccoli’s hairy root cultures via the mediation of Agrobacterium rhizogenes and investigates the effects of different concentrations of copper sulfate (CuSO₄) and zinc sulfate (ZnSO₄) as two elicitors on the production of sulforaphane in broccoli's hairy root. The expression of the Myrosinase (MY) gene, associated with sulforaphane production, was quantified using real-time PCR. Moreover, the cytotoxic effect of different treatments of sulforaphane on the breast cancer cell line, MDA-MB-231, was estimated and the transcription levels of apoptosis-associated genes were also determined. The highest amount of sulforaphane was produced at 4 µM CuSO₄ after 16 h and 16 µM ZnSO₄ after 8 h of elicitation. Overexpression of the MY gene was found to be associated with increased production of sulforaphane. All sulforaphane treatments induced a dose-dependent reduction in the viability of cancer cells. The sulforaphane extracted from broccoli's hairy roots treated with CuSO₄ exerted a higher cytotoxic effect on the MDA-MB-231 breast cancer cell line than the sulforaphane extracted from broccoli's hairy root treated with ZnSO₄. All treatments of sulforaphane up-regulated the Apoptosis Regulator (Bax) gene, Proapoptotic Bcl2 Associated X, Caspase-3, Caspase-8, and Caspase-9, while down-regulating the B-cell lymphoma 2 (Bcl-2) gene (antiapoptotic) transcription. The overall results showed an antiapoptotic effect of sulforaphane extract derived from broccoli's hairy roots on MDAMB-231 breast cancer cells, thereby establishing the potential anticancer activity of the sulforaphane produced in broccoli's hairy root cultures.