Figure 4.

Effects of parthenolide and artemisinin on doxorubicin accumulation and cytotoxicity (assessed as: LDH release, Trypan blue staining, percentage of apoptotic cells, percentage of cells in cycle). HT29 cells were cultured in the absence (CTRL) or in the presence of doxorubicin (5 µmol·L⁻¹, DOX) for 6 h, together with parthenolide (PART, 10 µmol·L⁻¹) or artemisinin (ART, 10 µmol·L⁻¹); when indicated, cells were pre-incubated for 1 h with BAPTA-AM (BAP, 10 µmol·L⁻¹), then washed in PBS before the incubation with the other drugs. (A) Doxorubicin intracellular accumulation (doxo) and release of LDH activity in the culture supernatant (LDH) were measured as described in Methods section. Measurements were performed in duplicate and data are presented as means ± SE (n = 4). The significance of DOX versus CTRL was P < 0.001 (not reported in the figure). Versus DOX: *P < 0.001. Versus DOX + PART or DOX + ART respectively: °P < 0.02. (B) Trypan blue staining (trypan) and FACS analysis of annexin V-FITC and PI-positive HT29 cells (annex/PI) were performed as reported in the Methods section. Measurements were performed in duplicate and data are presented as means ± SE (n = 3). The significance of DOX versus CTRL was P < 0.001 (not reported in the figure). Versus DOX: *P < 0.001. Versus DOX + PART or DOX + ART respectively: °P < 0.001. (C) Cells were incubated for 6 h as indicated above, then washed and grown for a further 24 h in fresh medium; after this time, cells were permeabilized and treated with PI, as described under Methods section. Cell cycle analysis of G₀/G₁ phase, S phase and G₂/M phase was obtained by the FACSCalibur system using the Cell Quest software (see Methods section for details). The measurements were performed in duplicate and data are presented as means ± SE (n = 3). Concerning the percentage of cells in S phase, the significance of DOX versus CTRL was P < 0.05 (not reported in the figure). Versus DOX: *P < 0.05. Versus DOX + PART or DOX + ART respectively: °P < 0.05. FITC, fluorescein isothiocyanate conjugate; LDH, lactate dehydrogenase; PBS, phosphate buffer saline; PI, propidium iodide.