Amygdalin

amygdalin from apricot kernels (Sigma-Aldrich, Taufkirchen, Germany) was freshly dissolved in tumor cell culture medium and then added to tumor cells at a concentration of 10 mg/ml for either 24 h or for 2 weeks [10] (link) to evaluate acute versus chronic treatment. Controls received tumor cell culture medium alone. In all experiments, treated tumor cell cultures were compared to the non-treated ones. To exclude toxic effects of amygdalin, cell viability was determined by trypan blue (Gibco/Invitrogen).

amygdalin from apricot kernels (Sigma-Aldrich, Taufkirchen, Germany) was freshly dissolved in cell culture medium and added to tumor cells in concentrations ranging from 1.25–10 mg/ml. Controls remained untreated. In all experiments, treated tumor cell cultures were compared to non-treated cultures. To assess toxic effects of amygdalin, cell viability was determined by trypan blue (Gibco/Invitrogen). For apoptosis evaluation the expression of Annexin V/propidium iodide (PI) was determined using the Annexin V-FITC Apoptosis Detection kit (BD Pharmingen, Heidelberg, Germany). Tumor cells were washed twice with PBS, and then incubated with 5 µl of Annexin V-FITC and 5 µl of PI in the dark for 15 min at RT. Cells were analyzed on a FACScalibur (BD Biosciences, Heidelberg, Germany). The percentage of apoptotic cells (early and late) in each quadrant was calculated using CellQuest software (BD Biosciences).

Resistant sublines were established by continuous exposure to increasing concentrations of the respective drugs, as described before [13 (link)]. The docetaxel-resistant tumor cells were exposed to 2.5 ng/mL docetaxel (Sanofi, Paris, France), and cabazitaxel-resistant tumor cells were exposed to 2.5 ng/mL cabazitaxel (Sanofi, Paris, France) three times a week.
amygdalin from apricot kernels (Sigma-Aldrich, Taufkirchen, Germany) was freshly dissolved in cell culture medium and added to tumor cells at 10 mg/mL based on earlier studies [14 (link)]. Controls remained untreated. In all experiments, treated tumor cell cultures were compared to nontreated cultures. To assess toxic effects of amygdalin and/or docetaxel and cabazitaxel, cell viability was determined by trypan blue (Gibco/Invitrogen).