Abstract
BackgroundBreast 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.MethodsHR+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.ResultsThe 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.ConclusionOverall, the Rib/Eve concurrent therapy augmented the in vitro cytotoxic effect, compared to Rib/Eve sequential therapy or single treatments.
Highlights
Breast cancer (BC) is the second most common type of cancer worldwide
The efficiency of Rb silencing, at 72 h post transfection was analysed by western blot analysis using an anti-human Rb Ab
The results indicate that both Rib and Eve, alone, markedly inhibited the viability of Rb wild type (WT) MCF-7 cells (Rib: IC50 at 48 and 72 h: 6 ± 0.5 and 4 ± 0.3 μg/ml (Fig. 1a and b), respectively; Eve: IC50 at 48 and 72 h: 10 ± 0.8 μg/ml and 8 ± 0.6 μg/ml (Fig. 1c and d), respectively)
Summary
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); resistance to CDK4/ 6 inhibitors frequently develops. HR+HER2− MCF-7 cells were silenced for Retinoblastoma (Rb) gene, and 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. The endocrine therapies, which target ER activity, are standard treatments for patients with ER+ and human epidermal growth factor receptor negative (HER2−) BC in both the early and the advanced/metastatic stages [1, 2]. Recent advances in elucidating the molecular mechanisms of crosstalk among ER, cell-cycle regulating proteins and intracellular signaling pathways, have provided the rationale for combining endocrine therapies with targeted agents [3]. The generation of non-selective CDK inhibitors failed due to combined lack of efficacy and excessive toxicity reported by clinical trials across different cancer types [5]
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