Herceptin Conjugate Induces Tumor Cell Necrosis tumor cell apoptosis [14,15,16]. To achieve Herceptin oligomers instead of a homodimers, we used a small crosslinker molecule, EDC, and deviated from the instructed time (2 h) and temperature (37uC) of the manufacturer when we generated the conjugates; we used a longer incubation time (4–6 h) at a lower temperature (room temperature). The conjugates contained three components with estimated molecular weights (MWs) of 148 kDa, 296 kDa, and 450 KDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the absence of the reducing agent dithiothreitol (DTT; Figure 1a, lane 2), suggesting that these proteins appeared in the conjugate may be an unconjugated wild- type antibody, a homodimer, and a homotrimer, respectively. Using another crosslinker, SMCC, we were able to generate the similar Herceptin oligomers as above by following the manufac- turer’s instruction (Figure 1a, lane 3). Suppression of tumor cell survival by the Herceptin conjugate treatment To evaluate the role of conjugated Herceptin via two independent linkers, both wild-type Herceptin and Herceptin conjugates (20 mg/mL) were incubated with two independent Her2-positive cell lines, colon cancer line OS187 and osteosar- coma line CCH.OS.D (Figure 1c). Interestingly, the EDC- Herceptin conjugates greatly inhibited survival of both types of tumor cells (Figure 1b), whereas the SMCC conjugates failed to so inhibit tumor cell survival even though the conjugation pattern was the same on SDS-PAGE (Figure 1a). This observation led us to use the EDC linker for the rest of our experiments described in this study. The reduction of tumor cell survival by the EDC-Herceptin conjugate was Her2-expression specific because no reduction of Figure 1. Suppression of Her2-positive tumor cell survival by EDC-Herceptin conjugate. (a) Gel analysis of the Herceptin conjugate. Analysis of Herceptin and Herceptin conjugates at 6% SDS-PAGE without reducing reagent DTT. Lane 1, wild-type Herceptin; lane 2, EDC-Herceptin conjugate; lane 3, SMCC-Herceptin conjugate; lane 4, molecular weight marker. (b) Reduction of Her2-positive tumor cell survival by EDC-Herceptin conjugate but not by SMCC-Herceptin conjugate. OS187 and CCH.OS.D cells were treated with the indicated molecules at 20 mg/mL for 72 h. MTT assay was used to determine the cell survival rate. (c) Her2 high expression cell lines OS187 and CCH.OS.D. Cells were stained with isotype control or PE-Her2 antibody. Her2 expression level was determined by using flow cytometry. Red dotted line, isotype control; blue line, Her2. *, P,0.05; **, P,0.01. doi:10.1371/journal.pone.0023270.g001 PLoS ONE | www.plosone.org 2 August 2011 | Volume 6 | Issue 8 | e23270