Fraction of cells undergoing apoptosis with various apoptosis inhibitors and rATG
Inhibitor Target . | zFK-fmk Negative control . | zVAD-fmk Pancaspase . | Pepstatin A Cathepsin D . | Genistein Tyrosine kinases . | zFA-fmk Cysteine proteases . | E-64d Cathepsins B + D . | SB203561 MAP kinase . |
|---|---|---|---|---|---|---|---|
| sBc | 97.9 ± 2.7 | 60.1 ± 3.2* | 98.7 ± 2.7 | 91.9 ± 3.1 | 82.4 ± 1.6 | 21.4 ± 0.5* | 96.3 ± 1.8 |
| RPMI-8226 | 97.9 ± 2.7 | 70.1 ± 11.9* | 92.2 ± 3.5 | 25.3 ± 2.9* | 100.7 ± 1.5 | 89.2 ± 2.8 | 97.5 ± 2.9 |
| U266 | 97.7 ± 4.0 | 64.4 ± 3.4* | 47.2 ± 1.8* | 97.2 ± 1.8 | 90.9 ± 4.6 | 95.2 ± 3.8 | 101.6 ± 1.8 |
| NCI-H929 | 100.0 ± 1.5 | 106.3 ± 0.7 | 20.9 ± 2.2* | 21.9 ± 1.7* | 111.8 ± 0.2 | 110.7 ± 0.6 | 106.0 ± 0.6 |
| ARH-77 | 83.9 ± 0.2 | 82.8 ± 0.2 | 8.6 ± 0.1* | 78.1 ± 0.5 | 84.5 ± 0.5 | 82.9 ± 0.1 | 86.3 ± 0.4 |
| HS-Sultan | 88.2 ± 5.3 | 86.1 ± 2.6 | 97.2 ± 5.3 | 80.7 ± 7.1 | 87.5 ± 7.7 | 57.2 ± 4.8* | 78.2 ± 2.1 |
| MC-CAR | 79.2 ± 0.9 | 83.2 ± 5.1 | 20.2 ± 3.9* | 81.2 ± 7.4 | 79.6 ± 9.1 | 89.5 ± 4.1 | 81.9 ± 6.7 |
| hMC | 89.1 ± 4.3 | 66.4 ± 7.1* | 54.1 ± 3.7* | 82.1 ± 6.8 | 88.2 ± 5.0 | 59.6 ± 5.1* | 83.4 ± 7.7 |
Inhibitor Target . | zFK-fmk Negative control . | zVAD-fmk Pancaspase . | Pepstatin A Cathepsin D . | Genistein Tyrosine kinases . | zFA-fmk Cysteine proteases . | E-64d Cathepsins B + D . | SB203561 MAP kinase . |
|---|---|---|---|---|---|---|---|
| sBc | 97.9 ± 2.7 | 60.1 ± 3.2* | 98.7 ± 2.7 | 91.9 ± 3.1 | 82.4 ± 1.6 | 21.4 ± 0.5* | 96.3 ± 1.8 |
| RPMI-8226 | 97.9 ± 2.7 | 70.1 ± 11.9* | 92.2 ± 3.5 | 25.3 ± 2.9* | 100.7 ± 1.5 | 89.2 ± 2.8 | 97.5 ± 2.9 |
| U266 | 97.7 ± 4.0 | 64.4 ± 3.4* | 47.2 ± 1.8* | 97.2 ± 1.8 | 90.9 ± 4.6 | 95.2 ± 3.8 | 101.6 ± 1.8 |
| NCI-H929 | 100.0 ± 1.5 | 106.3 ± 0.7 | 20.9 ± 2.2* | 21.9 ± 1.7* | 111.8 ± 0.2 | 110.7 ± 0.6 | 106.0 ± 0.6 |
| ARH-77 | 83.9 ± 0.2 | 82.8 ± 0.2 | 8.6 ± 0.1* | 78.1 ± 0.5 | 84.5 ± 0.5 | 82.9 ± 0.1 | 86.3 ± 0.4 |
| HS-Sultan | 88.2 ± 5.3 | 86.1 ± 2.6 | 97.2 ± 5.3 | 80.7 ± 7.1 | 87.5 ± 7.7 | 57.2 ± 4.8* | 78.2 ± 2.1 |
| MC-CAR | 79.2 ± 0.9 | 83.2 ± 5.1 | 20.2 ± 3.9* | 81.2 ± 7.4 | 79.6 ± 9.1 | 89.5 ± 4.1 | 81.9 ± 6.7 |
| hMC | 89.1 ± 4.3 | 66.4 ± 7.1* | 54.1 ± 3.7* | 82.1 ± 6.8 | 88.2 ± 5.0 | 59.6 ± 5.1* | 83.4 ± 7.7 |
rATG appears to activate both cathepsin- and caspase-mediated cell death pathways in myeloma cell lines and primary myeloma cells (hMCs), as well as tyrosine kinase–dependent apoptosis cascades. Myeloma cell lines were incubated with 250 μg/mL rATG in the presence of various inhibitors of apoptosis and then analyzed for cell death by TOPRO-3 staining. Results are the percentage of apoptotic cells in the test sample divided by the percentage of apoptotic cells in a control sample without any inhibitor. The control substance zFK-fmk was used as a second negative control. Each data point represents a mean of 3 independent experiments and results are means ± 1 SD.
Values with more than 20% decrease in rATG-induced cell death