Here we show for the first time that rLOX-PP protein inhibits pre-existing tumor growth after direct i.t. injection or when applied in a slow release formulation. Intratumoral injection studies have been classically done in initial studies to evaluate the ability of proteins to inhibit tumor growth. Direct i.t. injection of tumor necrosis factor-alpha and interferons, or interferons alone, were shown to inhibit xenograft growth in mice 
. In human patients, i.t. injection of recombinant IL-2 induced tumor killing, tumor necrosis and lymphocytic infiltration 
. Thus, consistent with other pre-clinical and clinical studies, it is here shown that direct i.t. injection of rLOX-PP inhibited the growth of a pre-existing breast cancer xenograft. Interestingly, the slow release formulation appeared to be more effective than i.t. injection of naked rLOX-PP in the inhibition of NF639 xenograft growth. Although, LOX-PP has been shown to have tumor suppressor properties, the effectiveness of rLOX-PP protein to inhibit pre-existing tumor growth has not been previously evaluated in vivo. Such studies are important in the evaluation of therapeutic potential.
Alginate beads have been used previously for successful slow release of recombinant proteins with a high isoelectric point without loss of biological activity 
. In human clinical studies, alginate beads containing human mature allogenic chondrocytes were found to be an effective treatment for cartilage defects in the knee showing significant improvement within six months which shows that alginate is safe and feasible for application in vivo in patients 
. Alginate formulations, therefore, could have potential for use in pre-clinical human studies, although other carriers such as polylactide/polyglycolide polymers may be more widely used 
. Encapsulated proteins in alginate beads are released by diffusion from and degradation of alginate beads for in vivo applications 
, and the present study is significant in that it provides proof of principle that rLOX-PP is effective in vivo, especially in a slow release formulation. At sacrifice, it was apparent that most of the bead material had been resorbed in all implanted mice, as expected. Only 1–2 beads out of the twelve beads implanted per mouse could be found at sacrifice (data not shown) suggesting that more than 80% of rLOX-PP was released in vivo.
The single implantation of alginate incorporated rLOX-PP is more efficient than five i.t. injections of rLOX-PP. The total amount of rLOX-PP incorporated in alginate beads was 35 micrograms and resulted in a more significant reduction in tumor xenograft volume, growth rate and tumor weight compared to 50 micrograms rLOX-PP direct i.t. injections over five days. Similarly, other studies have shown that alginate encapsulation of recombinant proteins are highly active in vivo. For example, similar to our data with rLOX-PP, in vivo subcutaneous implantation of a single dose of alginate beads containing IL-17R was more effective in reducing inflammation when compared to multiple subcutaneous injections of this anti-inflammatory protein 
. FGF-2 plays an important role in tissue repair and has a very short half-life when administered parenterally. Surgical implantation of FGF-2 bound to heparin-Sepharose beads encapsulated in calcium alginate microcapsules in an arterial injury model was more efficient in delivering FGF-2 within the arterial wall when compared to intravenous administration 
. Thus, consistent with other studies, rLOX-PP inhibits tumor xenograft by direct i.t. injection or implantation of alginate encapsulated rLOX-PP, yet the latter shows a stronger effect with an apparently increased persistence of rLOX-PP in tumors as seen in . Although there are many possibilities to improve the current formulation of rLOX-PP for in vivo application, the current formulation of alginate beads with encapsulated rLOX-PP inhibited tumor growth rate by more than 61% and provides proof of principle that rLOX-PP protein in some form has therapeutic potential. It now becomes of considerable interest to determine the pharmacokinetics, pharmacodynamics, and stability and effectiveness of systemically applied naked rLOX-PP and of protected formulations of rLOX-PP that would be acceptable for human trials in order to develop rLOX-PP as an anti-cancer drug candidate.
In order to investigate mechanisms of inhibition, we evaluated effects of rLOX-PP on cell proliferation and apoptosis. rLOX-PP inhibits cell proliferation determined by reduced expression of two independent proliferation markers: Ki-67 and phospho-histone H3. Ki-67 has been used to evaluate the responsiveness of chemotherapeutics to breast cancer patients and evaluate the risk of tumor incidence after treatments 
. Ki-67 has diagnostic importance in distinguishing between benign and malignant tumors 
, and is a prognostic factor for clinical breast cancer patients 
. Phospho-histone H3 has been similarly employed to evaluate tumor grade and aggressiveness 
. Phospho-histone H3 is a prognostic proliferation marker in triple negative invasive lymph node-negative breast cancer 
Data further show that rLOX-PP induces apoptosis in breast cancer xenografts which was evaluated by activated caspase 3 immunostaining and TUNEL assays. Apoptosis is induced by different pathways which converge at caspase 3 activation, a pro-apoptotic marker 
. Recently, it has been shown that rLOX-PP sensitizes pancreatic and breast cancer cells to doxorubicin induced apoptosis in vitro 
, but whether rLOX-PP alone could induce apoptosis in breast cancer xenografts in vivo was not known. The mechanism by which rLOX-PP induces apoptosis of cancer cells in vivo in the absence of other chemotherapeutic agents as seen in the present study requires further evaluations. The NCR nu/nu mouse model employed in the present study is T-cell deficient, but contains natural killer (NK) cells and B cells. It is, therefore, possible to speculate that the increased apoptosis observed in rLOX-PP treated tumors in vivo could be driven by enhanced sensitivity to NK cell-derived TRAIL, or FasL stimulated signaling 
. A greater understanding of molecular mechanisms of action of LOX-PP may permit the design of combinatorial therapeutic approaches that would be more effective than individual chemotherapeutic approaches. As noted, it is of interest that rLOX-PP inhibits tumor growth in vivo characterized by inhibition of cell proliferation and induction of apoptosis, whereas in vitro rLOX-PP alone inhibits proliferation but has not been seen to promote apoptosis 
Earlier in vitro studies done with ectopic rLOX-PP expression in cancer cell lines indicate that LOX-PP inhibits Ras-mediated activation of Erk1/2 by inhibiting FGF-2 to FGFR1 receptor binding and activation, and by direct interaction with Hsp70 and c-Raf 
. The current study extends these observations to rLOX-PP protein administered in vivo in which we show highly significant inhibition of Erk1/2 phosphorylation. It is likely that mechanisms of LOX-PP identified primarily in vitro are operative in vivo, but further study of individual molecular interactions in vivo with wild type and mutant forms of rLOX-PP will be required to investigate the relationship between specific molecular interactions and actual inhibition of tumor growth. The observation of similarities in the magnitudes of rLOX-PP administration on altering levels of both proliferation and apoptosis markers, whereas tumor inhibition is more pronounced in the slow release model is likely to be coincidental. Without more continuous assays of changes in the expression of these markers throughout the entire experimental period, it is difficult to comment further. Importantly, the current study establishes experimental systems by which a variety of questions can now be addressed in vivo.
In conclusion, rLOX-PP protein is effective in inhibiting mouse xenograft growth. The data show that alginate bound rLOX-PP is more efficient in inhibiting breast cancer xenograft compared to direct i.t injections. Further understanding of the mechanisms by which rLOX-PP inhibits breast cancer xenograft growth will enhance the ability to design potentially more effective combinations of anti-cancer regimens.