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D. J. Fitzgerald et al.


immunogenicity would allow more cycles of treat- ment. In solid tumor masses, immunotoxin delivery may be restricted because of tumor architecture and the presence of high concentrations of soluble target antigen. To address this, various combination therapies have been proposed to ‘break up’ tumor architecture, lowering the concentration of blocking antigen and delivering more immunotoxin [5]. Another possible reason for immunotoxin resistance relates to anti-apoptotic strategies frequently em- ployed by tumor cells to overcome chemotherapy- mediated death.With regard to the latter, high levels of prosurvival proteins such as Bcl-2, Bcl-xl, and Mcl-1 contribute to resistance. Because immunotox- in action frequently results in the loss of Mcl-1 (Mcl-1 is a short-lived protein and is not replenished when protein synthesis is shut down), we speculate that high levels of Bcl-2 or Bcl-xl could result in less than complete immunotoxin-mediated killing [6]. We are beginning to explore this concern.


Results and discussion


One way to combat a high expression level of prosurvival proteins is the use of a BH3-mimetic. To appreciate this approach, elements of the apoptosis machinery need to be considered. Activa- tion of the proapoptotic proteins, Bax and Bak, depends on the action of BH3-only proteins, which are up-regulated in response to stress or cell injury. BH3-only proteins can activate Bax/Bak directly or they can trigger apoptosis via the neutralization of the prosurvival proteins, Bcl-2, Bcl-xl, Mcl-1. Therefore, BH3 mimetics were developed as potential stand- alone antitumor agents or as sensitizers for che- motherapy, thus eliminating resistance to apoptosis. ABT-737, one such agent, was shown to have strong binding affinity for Bcl-2 and -xl but little or none for Mcl-1 [7]. Because immunotoxins result in the loss of Mcl-1 via inhibition of protein synthesis, it seemed worth investigating whether ABT-737 could sensitize cells to immunotoxin action. Therefore, experiments were conducted whereby cells were treated with immunotoxin alone, ABT-737 alone, or immuno- toxin plus ABT-737 [6]. We have completed this kind of experiment with several cell lines, including immunotoxin resistant cells, and frequently see a result qualitatively similar to that shown in Figure 1, where immunotoxin–ABT-737 combinations act synergistically to kill cells via apoptosis. Using caspase 3 activation as a measure of apoptosis, we note that DLD1 cells were not readily killed by either immunotoxin or ABT-737 alone. However, the combination showed extensive apoptotic cell death (Figure 1, and reportedmore extensively in reference [6]). Further, it should be noted that we have been


Figure 1. Immunotoxin resistance is overcome by combination treatment with ABT-737. DLD1 cells were treated with an immunotoxin (48 h) directed to the human transferrin receptor either alone or in combination with ABT-737. At 1 ng/mL there was no evidence of immunotoxin-mediated apoptosis as measured by activation of caspase 3. When the immunotoxin was added in combination with ABT-737, there was activation of caspase 3, indicating cell death via apoptosis. In data not shown, PARP [poly(ADP-ribose) polymerase] cleavage was also evident, but only in cells treated with the combination of immunotoxin and ABT- 737. ABT-737 was not toxic for DLD1 cells.


able to duplicate most of our findings obtained with ABT-737 with the related clinical compound, ABT- 263 (data not shown). In conclusion, our results support using combina-


tion treatments whereby immunotoxins are adminis- tered along with agents that overcome resistance. These adjunct agents will likely be chosen from compounds that either reduce antibody responses, increase tumor ‘break up,’ or sensitize cells to apoptosis. While laboratory experiments point the way for such development, concepts will have to be evaluated and treatments proven in the clinic.


Potential conflict of interest: Disclosure forms provided by the authors are available with the full text of this article at www.informahealthcare.com/lal.


References


1. Pastan I, Hassan R, FitzGerald DJ, Kreitman RJ. Immunotoxin treatment of cancer. Annu Rev Med 2007;58:221–237.


2. Du X, Youle RJ, FitzGerald DJ, Pastan I. Pseudomonas exotoxin A-mediated apoptosis is Bak dependent and preceded by the degradation of Mcl-1. Mol Cell Biol 2010;30: 3444–3452.


3. Andersson Y, Juell S, Fodstad Ø. Downregulation of the antiapoptotic MCL-1 protein and apoptosis in MA-11 breast cancer cells induced by an anti-epidermal growth factor receptor-Pseudomonas exotoxin a immunotoxin. Int J Cancer 2004;112:475–483.


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