Dihydroconiferyl Ferulate Modulates Breast Cancer Stemness

MDA-MB-231 cancer cells (1.5 × 10⁶ cells) were cultured in a 6-well plate for 24 h and treated with DMSO as control or dihydroconiferyl ferulate (50 μM) for 24 h [63 (link)]. The cells were harvested, dissociated, and incubated with monoclonal antibody antihuman CD44 (FITC-conjugated) and monoclonal antibody antihuman CD24 (APC-conjugated) antibodies (BD) for 45 min at 4 °C. After washing with 1X PBS, the CD44⁺/CD24⁻ cell populations were examined using an Accuri C6 machine (BD San Jose, CA, USA). The Annexin V Apoptosis Detection kit with PI (BD) was used to measure apoptosis of the mammospheres treated with dihydroconiferyl ferulate (50 μM) following the manufacturer’s protocol. Mammospheres were collected and dissociated with 0.05% trypsin-EDTA 1X (Corning). Briefly, 1 × 10⁶ cells were incubated with Annexin V (FITC) and PI in a binding buffer at room temperature (RT), protected from light for 30 min, and the cells were examined via flow cytometry at the Jeju Center of Korea Basic Science Institute (core facility center, Jeju, South Korea). Mammospheres derived from MDA-MB-231 cells cultured with or without dihydroconiferyl ferulate (50 µM) were divided into single cells, and an equal number of cancer cells were seeded into six-well plates. The number of cells was counted at 24, 48, and 72 h to measure the growth of the mammospheres.