Herceptin Conjugate Induces Tumor Cell Necrosis Figure 7. Identification of the primary conjugate fraction for triggering a significant amount of tumor cell death. (a) HPLC separation profile of the EDC-Herceptin conjugates. The dimer and trimer were estimated from the MWs of running wild-type Herceptin and the standard proteins. (b) Coomassie blue staining of separated EDC-Herceptin conjugate. 1, monomer; 2, dimer; 3, trimer. (c) Cell survival comparison following the treatments with Herceptin monomer, dimer, and trimer of the EDC-linked Herceptin (20 mg/mL). MTT assay was used. *, P,0.05; **, P,0.01; ***, P,0.001. doi:10.1371/journal.pone.0023270.g007 inhibitor. Neither of the caspase inhibitors could reverse EDC Herceptin conjugate induced tumor cell death (Figure 6b), which suggested apoptosis didn’t cause such cell death. Induction of RIP receptor-interacting protein-dependent PNCD by Herceptin conjugate treatment Late apoptotic cells were positively stained for both apoptotic and necrosis markers. It would be interesting to know whether necrosis contributes to the EDC-Herceptin conjugate-induced cell death. Necrotic cell death occurs in two ways: (1) acute unprogrammed necrotic death and (2) PNCD. We were solely interested in detecting the PNCD because unprogrammed necrotic cell death typically causes an indiscriminate and massive cell death in an acute time period, which was not observed in our treatment. Our EDC-Herceptin conjugate induced solely Her2- specific tumor cell death. To test which cell death mechanism accounted for the EDC-Herceptin conjugate-induced cell death, a necrosis inhibitor was used to definitively determine that necrosis accounted for the Herceptin conjugate-mediated cell death. Necrostatin-1 (Nec-1), a potent and specific inhibitor for PNCD, was used because this inhibitor perturbs the key PNCD-regulator protein, called the receptor-interacting protein (RIP) [23]. The result confirmed our hypothesis because Nec-1 treatment reversed the cell survival reduction by Herceptin conjugate treatment as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo- lium bromide (MTT) assay (Figure 6c), which suggested that necrosis was the crucial mechanism in EDC-Herceptin conjugate- induced Her2-positive tumor cell death. The critical component among the Herceptin conjugates for inducing direct tumor cell death The EDC-Herceptin conjugates contained three different proteins: wild-type antibody (referred to as monomer), the fusion of two wild-type antibody (referred to as dimer), and the fusion of three antibody molecules (referred to as trimer) (Figure 1). The question was which form of the conjugate played the most significant role in inducing cell death. To this end, the mixture of the Herceptin conjugates was separated using a high-performance liquid chromatography (HPLC) system, and major peaks were found at the estimated retention times of 15, 16, and 17 min (Figure 7a). Based on the retention time of standard proteins with different MWs and wild-type antibodies run on the same HPLC separation system, it was determined that the fractions collected from the shorter retention time (14–15 min) contained a conjugate with an estimated MW of 450 kDa, which was the trimer antibody; fractions collected from the longer retention time PLoS ONE | www.plosone.org 8 August 2011 | Volume 6 | Issue 8 | e23270