Breast cancer (BC) is the second most common type of cancer worldwide. Among targeted therapies for Hormone Receptor-positive (HR⁺) and Human Epidermal growth factor Receptor 2-negative (HER2⁻) BC, the Cyclin-Dependent Kinases (CDK4/6) are targeted by inhibitors such as Ribociclib (Rib); however, resistance to CDK4/6 inhibitors frequently develops. The aim of this work is to assess in vitro activity of Rib and Everolimus (Eve) in HR⁺HER2⁻ MCF-7 and HR⁻HER2⁻BT-549 BC cell lines.

HR⁺HER2⁻ MCF-7 and HR⁻HER2⁻ BT-549 BC cell lines were treated with increasing concentration of Rib and Eve (up to 80 μg/mL) for 48–72 h. Subsequently, HR⁺HER2⁻ MCF-7 cells were silenced for Retinoblastoma (Rb) gene, and thus, the effect of Rib in sequential or concurrent schedule with Eve for the treatment of both Rb wild type or Rb knock-down MCF-7 in vitro was evaluated. Cell viability of HR⁺HER2⁻ MCF-7cells treated with sequential and concurrent dosing schedule was analyzed by MTT assay. Moreover, cell cycle phases, cell death and senescence were evaluated by cytofluorimetric analysis after treatment with Rib or Eve alone or in combination.

The sequential treatment didn’t produce a significant increase of cytotoxicity, compared to Rib alone. Instead, the cotreatment synergized to increase the cytotoxicity compared to Rib alone. The cotreatment reduced the percentage of cells in S and G2/M phases and induced apoptosis. Rib triggered senescence and Eve completely reversed this effect in Rb wild type BC cells. Rib also showed Rb-independent effects as shown by results in Rb knock-down MCF-7.

Overall, the Rib/Eve concurrent therapy augmented the in vitro cytotoxic effect, compared to Rib/Eve sequential therapy or single treatments.