Herceptin Conjugate Induces Tumor Cell Necrosis Figure 4. Induction of tumor cell death by EDC-Herceptin conjugate treatment. (a) BrdU incorporation rate following different treatments. Cells were treated for 72 h as described in Figure 2, followed by BrdU flow assay using flow cytometry. (b) Live and dead cell analysis on OS187 cells after treatment, as described in Figure 3. doi:10.1371/journal.pone.0023270.g004 Induction of necrosis-mediated tumor cell death and induction of late-stage apoptosis account for cell death induced by the EDC-Herceptin conjugates Our results clearly indicated that the EDC-Herceptin conjugate significantly (P,0.05) induced Her2-positive tumor cell death, but the mechanism was unknown. Apoptosis is the most common cell death mechanism, and the origins of Herceptin conjugate-induced cell death could be attributed to the induction of apoptosis. To elucidate the mechanism of the cell death induction by the EDC- Herceptin conjugate, we chose an EDC-Herceptin conjugate treatment sensitive cell line (OS187) and a less sensitive cell line (SKBR3) for the following study. To determine whether the apoptotic activity truly and solely accounted for the Herceptin conjugate-mediated tumor cell death, we used flow cytometry to compare the difference in cell death (both necrosis and apoptosis) between samples treated with wild- type Herceptin and samples treated with Herceptin conjugates. To detect the percentage of apoptotic cells and necrotic cells in the different antibody treatments, the treated cells were stained with fluorescein isothiocyanate (FITC)-annexin V and propidium iodide (PI) [20]. The cell population with both annexin V and PI negative represented viable cells. Cells stained with both annexin V and PI positive represented late apoptotic cells. The cell population with annexin V positive but PI negative represented early stage apoptotic cells. The cell population with PI positive but annexin V negative represented dead cells. In the OS187 cells, we discovered 48 h after treatment, compared with sham and wild- type Herceptin, the EDC conjugate induced a slight increase in the dead cell population (Figure 5a). 72 h after treatment, wild- type Herceptin-treated cells increased early and late stage apoptosis, while the EDC-Herceptin conjugate induced more than two times the number of dead cells compared with the sham and wild-type Herceptin (Figure 5a). In our necrosis control, we froze OS187 cells at 280uC for 5 min and then thawed the cells at 37uC. The severe freeze-thaw process devastated most cells so that cells tended to become necrotic. When we performed a PI-annexin V assay, we found EDC-Herceptin conjugate-treated cells had a trend similar to that of the necrosis control cells, suggesting that induction of necrosis might account for the EDC-Herceptin conjugate’s effect. The SKBR3 cells were less sensitive to the EDC-Herceptin conjugate treatment. This result showed that, at a low dose (20 mg/mL), neither the wild-type Herceptin nor the EDC conjugate had much effect. However, high doses of the EDC-Herceptin conjugate greatly boosted the late apoptosis and necrosis cell population without any increase in early stage apoptosis (Figure 5b). Such results suggested that late apoptosis and necrosis might account for the EDC-Herceptin conjugate’s effect on both cells lines. The difference of PI-annexin V staining between the two cell lines also explained the sensitivity difference of these two cells lines to EDC conjugate treatment. All the flow cytometry experiments were repeated at least three times with the same or similar results. Induction of apoptotic enzyme activities by EDC- Herceptin conjugates Our results indicated that apoptosis might contribute to the EDC conjugate-mediated Her2-positive tumor cell death. Apop- tosis involves two major activation pathways [21,22]: (1) internal via mitochondrial transmembrane stress and (2) external via death ligand binding activity. The former activates initiator caspase-9, and the latter activates caspase-8. Both caspase-9 and caspase-8 can activate apoptotic executor caspase-3. To determine the caspase pathway that could contribute to the EDC conjugate- mediated tumor cell death, the activated caspases 3, 8, and 9 were PLoS ONE | www.plosone.org 5 August 2011 | Volume 6 | Issue 8 | e23270