Metronomic combination of Vinorelbine and 5Fluorouracil is able to inhibit triple-negative breast cancer cells. Results from the proof-of-concept VICTOR-0 study

Abstract

Triple Negative Breast Cancer (TNBC) is an aggressive neoplasia with median Overall Survival (OS) less than two years. Despite the availability of new drugs, the chance of survival of these patients did not increase. The combination of low doses of drugs in a metronomic schedule showed efficacy in clinical trials, exhibiting an anti-proliferative and anti-tumour activity. In Victor-2 study we recently evaluated a new metronomic combination (mCHT) of Capecitabine (CAPE) and Vinorelbine (VNR) in breast cancer patients showing a disease control rate with a median Progression-Free Survival (PFS) of 4.7 months in 28 TNBC patients. Here in Victor-0 study, we examined the effect of mCHT vs standard (STD) schedule of administration of different combinations of 5-Fluorouracil (5FU), the active metabolite of CAPE, and VNR in TNBC cell lines MDA-MB-231 and BT-549. A significant anti-proliferative activity was observed in cells treated with metronomic vs STD administration of 5FU or VNR alone. Combination of the two drugs showed an additive inhibitor effect on cell growth in both cell lines. Moreover, after exposure of cells to 5FU and VNR under mCHT or conventional schedule of administration we also observed a downregulation of chemoresistance factor Bcl-2, changes in pro-apoptotic protein Bax and in cleaved effector caspase-3 and increased expression of LC3A/B autophagy protein. Our results therefore suggest that molecular mechanisms implicated in apoptosis and autophagy as well as the cross-talk between these two forms of cell death in MDA-MB-231 and BT-549 cells treated with 5FU and VNR is dose- and schedule-dependent and provide some insights about the roles of autophagy and senescence in 5FU/VNR-induced cell death.

Keywords: 5-fluorouracil; LC3A/B; metronomic combination; triple-negative breast cancer; vinorelbine.

Figure 1. Metronomic administration of 5FU and VNR induced significant growth inhibition in human MDA-MB-231 and BT-549 breast cancer cells

(A) MDA-MB-231 and (B) BT-549 cells were treated with different concentration of 5FU and VNR for 4 h (STD) or 96 h (mCHT). The dose-response curves of MDA-MB-231 (C) and BT-549 (D) were used to calculated IC₅₀ value. Cell viability was investigated using the MTT assay and expressed as a percentage relative to the untreated control cells. The experiment was repeated 4 times with at least 8 replicates per sample. ^*p < 0.05 vs untreated ^**p < 0.01 vs untreated. Results are means ± SD of three measurements (P < 0.05).

Figure 2. Metronomic administration of 5FU and VNR in combination induced significant growth inhibition in human MDA-MB-231 and BT-549 breast cancer cells

Representative dose-response curve performed on MDA-MB-231 (A) and BT-549 (B) treated with the following drug combination: 1) 2x IC₅₀ (5FU) + 2x IC₅₀ (VNR) 2) IC₅₀ (5FU) + IC₅₀ (VNR) 3) ½ IC₅₀ (5FU) + ½ IC₅₀ (VNR); 4) ¼ IC₅₀ (5FU) + ¼ IC₅₀ (VNR); cells were treated for 4 h (STD) or 96 h (mCHT) and their number evaluated by MTT assay. The reading values were converted to the percentage and compared to untreated control. The simple two-point method uses 2 data points bracketing 50% inhibition of proliferation (red lines) to estimate the IC₅₀. The experiment was repeated 3 times with at least 8 replicates per sample.

Figure 3. 5FU and VNR can induce either apoptosis and/or autophagy in TNBC cells depending on the schedule of their administration

(A) Upper panel: representative Western blot of MDA-MB-231 and BT-549 exposed to 5FU and VNR alone (IC₅₀ single drug) or in combination (IC₅₀ combo) for 4 h (STD) and for 96 h (mCHT). (B) Quantification of the protein expression as evaluated by densitometry. Protein levels were normalized to the corresponding Vinculin loading control. Error bars represent mean ± SEM, n = 3.

Figure 4. Increased of autophagy response in MDA-MB-231 and BT-549 cells treated with 5FU and VNR in metronomic schedule

(A) MDA-MB-231 and (B) BT-549 cells were exposed to 5FU and VNR alone (IC₅₀ single drug) or in combination (IC₅₀ combo) for 4 h (STD) and for 96 h (mCHT). Anti-LC3A/B was detected by a FITC-conjugated secondary antibody, TRITC-conjugated phalloidin was used to stain actin and nuclei were counterstained with DAPI before acquiring images by confocal microscope (Biorad Laboratories, Hercules CA, USA). White arrows indicate autophagosomes fully formed in the cytoplasm of the cells upon exposure to VNR alone or in combination with 5FU in metronomic schedule. The Yellow arrows indicate apoptotic cells that are shrunken with condensed cytoplasm.