Figure 6.
Figure 6. Bortezomib induces mitochondrial transmembrane potential loss, apoptosis, and caspase 3 activation in CTCL cells. SeAx, MyLa, and PBLs from patients with Sézary syndrome were treated with increasing concentrations (5-40 nM) of bortezomib for 24 to 48 hours. (A) Annexin-V binding was carried out with an Annexin V–FITC detection kit and measured by flow cytometry. Abscissa and vertical axis represent the fluorescent intensities of annexin V–FITC and PI, respectively. For each panel, the numbers in bottom right and top right quadrants indicate the percentages of annexin V+/PI– cells (apoptotic, viable cells) and annexin V+/PI+ cells (dead cells by apoptosis), respectively. The mitochondrial transmembrane potential Δψm was detected using the fluorescent probe DiOC6 by flow cytometry. The numbers indicate the percentages of cells exhibiting a decrease in Δψm. The overlaying ashed curve represents the control untreated cells. The results presented for SeAx cells (top panel) are representative of 1 of 6 independent experiments. Results obtained with PBLs from one patient with Sézary syndrome (bottom panel) are representative of data derived from 12 patients (panel B). (B) PBLs from 12 patients with Sézary syndrome and from 8 healthy donors were treated with increasing concentrations (10-40 nM) of bortezomib for 24 and 48 hours (left and right panels, respectively) and assayed for apoptosis by detection of Δψm alterations. The percentages of low Δψm cells were determined in duplicate and normalized using control untreated cells, which were arbitrarily set as 100. Results obtained with PBLs from healthy donors and from SS patients are represented by □ and ▪, respectively. Each value represents the mean ± SEM (n = 12 and n = 8, SS and healthy PBLs, respectively). *Statistically significant difference between healthy and SS PBLs. (C) CTCL cell lines (SeAx and MyLa) and PBLs from 2 patients with Sézary syndrome were treated with increasing doses of bortezomib (10-40 nM) for 48 hours. Cells were analyzed by Western blotting for expression of pro-caspase 3 (32 kDa) and PARP as an intact (116-kDa) or cleaved (85-kDa) protein. GAPDH signal was used as an internal control for loading. Data represent 1 of 2 independent experiments giving similar results.

Bortezomib induces mitochondrial transmembrane potential loss, apoptosis, and caspase 3 activation in CTCL cells. SeAx, MyLa, and PBLs from patients with Sézary syndrome were treated with increasing concentrations (5-40 nM) of bortezomib for 24 to 48 hours. (A) Annexin-V binding was carried out with an Annexin V–FITC detection kit and measured by flow cytometry. Abscissa and vertical axis represent the fluorescent intensities of annexin V–FITC and PI, respectively. For each panel, the numbers in bottom right and top right quadrants indicate the percentages of annexin V+/PI– cells (apoptotic, viable cells) and annexin V+/PI+ cells (dead cells by apoptosis), respectively. The mitochondrial transmembrane potential Δψm was detected using the fluorescent probe DiOC6 by flow cytometry. The numbers indicate the percentages of cells exhibiting a decrease in Δψm. The overlaying ashed curve represents the control untreated cells. The results presented for SeAx cells (top panel) are representative of 1 of 6 independent experiments. Results obtained with PBLs from one patient with Sézary syndrome (bottom panel) are representative of data derived from 12 patients (panel B). (B) PBLs from 12 patients with Sézary syndrome and from 8 healthy donors were treated with increasing concentrations (10-40 nM) of bortezomib for 24 and 48 hours (left and right panels, respectively) and assayed for apoptosis by detection of Δψm alterations. The percentages of low Δψm cells were determined in duplicate and normalized using control untreated cells, which were arbitrarily set as 100. Results obtained with PBLs from healthy donors and from SS patients are represented by □ and ▪, respectively. Each value represents the mean ± SEM (n = 12 and n = 8, SS and healthy PBLs, respectively). *Statistically significant difference between healthy and SS PBLs. (C) CTCL cell lines (SeAx and MyLa) and PBLs from 2 patients with Sézary syndrome were treated with increasing doses of bortezomib (10-40 nM) for 48 hours. Cells were analyzed by Western blotting for expression of pro-caspase 3 (32 kDa) and PARP as an intact (116-kDa) or cleaved (85-kDa) protein. GAPDH signal was used as an internal control for loading. Data represent 1 of 2 independent experiments giving similar results.

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