(16 min) contained a conjugate with an estimated MW of 300 kDa, which was the dimer; and the fractions collected from the longest retention time of 17 min contained the monomer with an estimated MW of 150 kDa. Coomassie blue staining of the separated EDC-Herceptin conjugates verified the three conjugate components (Figure 7b). We noticed that these three components could not be completely isolated by HPLC. However, the key component (monomer, dimer, or trimer) was at least 50% of the total amount of proteins in each purified fraction, which was not perfect but was good enough for us to determine the effect of each individual protein. Treatment of OS187 and SKBR3 tumor cells with either purified Herceptin trimer or dimer, but especially the trimer, significantly increased tumor cell death compared with the wild-type antibody (Figure 7c). The EDC-Herceptin conjugated trimer was proved to be the most effective component in cell death induction. This dimer- or trimer-induced cell death was Her2- expression specific because no inhibition of cell viability was observed in the Her2-negative Col cell line. These results clearly suggested that the Herceptin trimer was the primary driving force causing PNCD. Discussion Our results showed that only EDC-Herceptin conjugates promoted direct tumor cell death, while the SMCC conjugates failed to affect Her2-positive cell viability, which is different from the previous report [8]. This discrepancy in the SMCC conjugate result between us and the previous report from Ghetie et al. (1997) is perhaps due to a difference in conjugate conditions. In Ghetie et al. study, the monoclonal Her2 antibody Her-50 instead of Herceptin and two crosslinkers, SMCC and N-succinimidyl S- acetylthioacetate (SATA), were used to generate the homodimer. The homodimer HER-50 was reported to increase cell death via an apoptotic mechanism [8]. In our study, a low temperature (room temperature) and longer incubation time were employed. Our EDC conjugate induced PNCD. It is also possible that the EDC conjugate is more effective than the SMCC conjugate because the former generates a bond between the N-terminal from one antibody molecule and the C-terminal of another antibody molecule to form the dimer or trimer. As a result of this nature, the dimer or trimer is relatively relaxed by the EDC linker as compared with the SMCC linker, which yields thioether bonds by possibly using the cysteine in the hinge region of the antibody. The relaxed antibody has a longer extension to bind on different cells with the same conjugate, causing cell death. Our study reveals for the first time that Herceptin, when forming a dimer or trimer via EDC, is capable of inducing PNCD, also called necroptosis. Necroptosis occurs naturally in embryonic development in addition to adult tissue homeostasis. It can be induced by tumor necrosis factor receptor (TNFR) activation, toll- like receptor (TLR) signaling, cytokines, and other factors. RIP kinase 1 serine/threonine kinase activity is required for death factor-mediated necroptosis [24] (Figure 5). It is clear that PNCD is part of the mechanism by which the Herceptin dimer or trimer induces tumor cell death, but the details remain unclear. One possible model explaining the Herceptin trimer antibody-induced PNCD is that the Herceptin trimers bind multiple antigens in the same cell, changing the stability of the membrane. This model is supported by the fact that only IgM therapy induces direct tumor cell death and a therapeutic effect [14,15,16,25]. Another possibility is that a single molecule binds to Her2 from different tumor cells, causing cell death. This theory is supported by the fact that IgM antibody against a B104 cell line illustrated a necrosis-like death [26]. Herceptin Conjugate Induces Tumor Cell Necrosis To improve the potency of Herceptin therapy, it is critical to understand the mechanism responsible for antibody-induced tumor cell death and from where the resistance originates. Multiple mechanisms contribute to tumor cell resistance to Herceptin [19,27,28]. These resistant mechanisms were summa- rized into two categories by Liu’s group [19]: (1) inhibition of antibody binding to the cognate receptors owing to truncation or the formation of homodimers and heterotrimers [19,29,30,31] and (2) activation of Herceptin-resistant signaling pathways, such as activation of the phosphoinositide 3-kinase [32], and inactivation of Herceptin Herceptin-sensitive pathways, such as downregula- tion of p27kip1 and phosphatase and tensin homolog (PTEN) protein [19,33]. Obviously, to overcome the tumor resistance to Herceptin, it is critical to overcome the binding resistance, the Herceptin-resistant signaling, or both. Huang et al. (2010) has found that Her2, human epidermal growth factor receptor 3 (Her3), and insulin-like growth factor 1 receptor (IGF-1R) formed a heterotrimeric complex in HR20 cells, which will likely cause conformational change of the Her2 receptor. Thus, EDC- Herceptin conjugates, particularly the Herceptin trimer, may have a better binding efficiency than wild-type Herceptin to the Her2 receptor in the heterotrimeric complex of Her2/Her3/IGF- 1R. The published results also indicate that antibody modifications and antibodies combined with standard treatments could increase target receptor internalization, arresting tumor cell growth or enhancing tumor cell death [8,23,34,35,36,37]. Our study illustrates an independent approach and mechanism to overcome the tumor resistance to antibody treatment and to induce necrotic cancer cell death. The induction of necroptosis has great potential to treat apoptosis-resistant cancer cells. Materials and Methods Ethics Statement There are no human participants or animal work involved in this study. Cell culture and establishment of Herceptin resistant cells The cancer cell lines SKBR3 and MDA-MB-468 were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). The OS187 [38], CCH.OS.D, and Col [38] cells were provided by Dr Dennis PM Hughes’s laboratory (The University of Texas MD Anderson Cancer Center, Houston, TX, USA). The CCH.OS.D is an osteosarcoma cell line derived from patients at the Children’s Cancer Hospital at The University of Texas M.D. Anderson Cancer Center. Fingerprint analysis recently defined the OS187 colon cancer line a subline of HCT15 and the Col neuroblastoma line. HCT15 is available from the American Type Culture Collection (ATCC, Rockville, MD, USA). Herceptin- resistant cells HR20 were obtained as described by Dr Bolin Liu [19]. Cell lines were grown in Dulbecco’s modified Eagle’s medium (Mediatech, Inc., Manassas, VA, USA) supplemented with glutamine and heat-inactivated 10% fetal calf serum (FCS) and 10 U/mL penicillin and streptomycin. Herceptin conjugate generation Herceptin was purchased from Genentech, Inc. (San Francisco, CA, USA). EDC (Pierce, Rockford, IL, USA) was the zero-length crosslinking agent used to couple carboxyl groups to primary amines. A quantity of 2 mg EDC was dissolved in 200 mL water and immediately mixed with 5 mg Herceptin, which was contained in the 400 mL reaction buffer of 0.1 M 2-(N- morpholino)-ethane sulfonic acid (pH 4.5) and incubated at room temperature for 4 h. Excess EDC and by-product were removed PLoS ONE | www.plosone.org 9 August 2011 | Volume 6 | Issue 8 | e23270