Bortezomib induced apoptosis in the neoplastic NK cells. (A) The NK lymphoma (NK-YS) and NK leukemia (KHYG-1, YT and NK-92) cell lines and short-term primary cultures from tumor biopsies of 2 patients with ENKL were treated with 0.5 to 150 ng/mL bortezomib for 24 hours. Viable cells were measured in triplicate with the MTS assay (Promega, Madison, WI), and results are presented as relative absorbance equal to the percentage of the average reading in untreated cells (±1 standard deviation). (B) After NK-cell lines were treated with 15 ng/mL bortezomib for 24 hours, the cells were collected and stained immediately with 5 μg/mL PI (Sigma, St Louis, MO) to distinguish viable cells from nonviable cells. The fluorescence was measured using the flow cytometer FACSCalibur (BD Biosciences, San Jose, CA). Data were analyzed by the WinMDI v2.8 software (Joseph Trotter, http://facs.scripps.edu/). Histograms of PI fluorescence were overlaid to show the altered distribution between before (thin line) and after (thick line) incubation with bortezomib. Because the dead cells are permeable to PI and thus stainable by PI, they correspond to the population with high fluorescence signals in the chart (arrows). (C) After treatment with 5 ng/mL bortezomib, KHYG-1 cells were stained with Annexin V/PI (BD Biosciences) and examined cytometrically. A population of apoptotic cells appeared in 6 hours with the Annexin V+/PI− phenotype. (D) After treatment with 15 ng/mL bortezomib, neoplastic NK cells were collected at different times and stained with Mitotracker Red (Molecular Probes, Eugene, OR) for Δmψ detection with flow cytometry. Histograms were overlaid to show the altered distribution (arrows) between the time before (thin line) and after 9-hour incubations with bortezomib (thick line). (E) Bortezomib inhibited YT cells in vivo. Four-week-old nude mice (n = 20) were injected subcutaneously in a single flank with 4 × 106 YT cells. Implanted tumors successfully engrafted in 10 mice. The tumor-bearing animals were injected intraperitoneally with bortezomib (n = 5) at 1 mg/kg twice a week or with vehicle only (n = 5). The subcutaneous tumor was measured with a caliper, and tumor size was calculated by the following formula: volume = 0.166 × π × length × width.2 The experiment lasted for 2 weeks until the animals were killed due to big tumor size (< 20 mm) in mock-treated mice. Means (±1 standard deviation) are shown. We compared the data by t test and found the result statistically significant (P = .001).
