Recombinant Lysyl Oxidase Propeptide Protein Inhibits Growth and Promotes Apoptosis of Pre-Existing Murine Breast Cancer Xenografts

Lysyl oxidase propeptide (LOX-PP) ectopic overexpression inhibits the growth of cancer xenografts. Here the ability and mode of action of purified recombinant LOX-PP (rLOX-PP) protein to inhibit the growth of pre-existing xenografts was determined. Experimental approaches employed were direct intratumoral injection (i.t.) of rLOX-PP protein into murine breast cancer NF639 xenografts, and application of a slow release formulation of rLOX-PP implanted adjacent to tumors in NCR nu/nu mice (n = 10). Tumors were monitored for growth, and after sacrifice were subjected to immunohistochemical and Western blot analyses for several markers of proliferation, apoptosis, and for rLOX-PP itself. Direct i.t. injection of rLOX-PP significantly reduced tumor volume on days 20, 22 and 25 and tumor weight at harvest on day 25 by 30% compared to control. Implantation of beads preloaded with 35 micrograms rLOX-PP (n = 10) in vivo reduced tumor volume and weight at sacrifice when compared to empty beads (p<0.05). A 30% reduction of tumor volume on days 22 and 25 (p<0.05) and final tumor weight on day 25 (p<0.05) were observed with a reduced tumor growth rate of 60% after implantation. rLOX-PP significantly reduced the expression of proliferation markers and Erk1/2 MAP kinase activation, while prominent increases in apoptosis markers were observed. rLOX-PP was detected by immunohistochemistry in harvested rLOX-PP tumors, but not in controls. Data provide pre-clinical findings that support proof of principle for the therapeutic anti-cancer potential of rLOX-PP protein formulations

Apoptosis marker active caspase 3 is increased by rLOX-PP treatment.

<p>(A) Immunostaining of tumors with active caspase 3 antibody, or non-immune IgG control. (A) i.t. rLOX-PP- or PBS injected tumors, and (B) tumors treated with surgically implanted empty alginate beads or rLOX-PP/alginate beads. Black arrows mark some positive-stained cells; the white arrows point to an inset containing an enlarged image of a stained cell. Scale bar = 0.005 mm for 400× and 0.002 mm for 1000×; (C) the corresponding quantifications (***, p<0.001; n = 3).</p

rLOX-PP persists in treated tumors.

<p>Immunostaining of tumors after sacrifice with LOX-PP antibody or non-immune IgG control from mice after (A) direct i.t. injection of rLOX-PP compared to PBS or (B) after implantation of empty or rLOX-PP-containing alginate beads. Scale bars = 0.005 mm. Arrows mark some positive-stained cells.</p

Alginate bead release kinetics of rLOX-PP.

<p>(A) Western blot of known amounts of rLOX-PP ranging from 0.1 to 10 ng of rLOX-PP; (B) Western blot of rLOX-PP-bead supernatants collected at different intervals; (C) calculated cumulative release of rLOX-PP; (n = 4; *, p<0.001).</p

Proliferation marker phosphorylated histone H3 is decreased by rLOX-PP treatment.

<p>(A) Immunostaining with phospho- histone H3 antibody or nonimmune IgG control tumors after (A) direct i.t. injection of rLOX-PP (L) compared to PBS (P) and (B) after implantation of empty (EB) and rLOX-PP (LB) alginate beads. Black arrows mark some positive-stained cells; the white arrows point to an inset containing an enlarged image of a stained cell. Scale bars correspond to 0.005 mm for 400× and 0.002 mm for 1000×. Arrows mark some positively stained cells. (C) Western blot analyses of 17 kDa phospho- histone H3, 36 kDa beta-actin and the corresponding quantifications of protein extracts from tumors after (C) direct i.t. injections of rLOX-PP compared to PBS and (D) after implantation of empty and rLOX-PP alginate beads (*, p<0.01; n = 3).</p

Proliferation marker Ki-67 is decreased by rLOX-PP treatment.

<p>(A) Immunostaining with Ki-67 antibody or non-immune IgG control tumors after (A) direct i.t. injection of rLOX-PP compared to PBS and (B) after implantation of empty and rLOX-PP alginate beads. Black arrows mark some positive-stained cells; the white arrows point to an inset containing an enlarged image of a stained cell. (C) The corresponding quantifications are shown (***, p<0.001; n = 3). Scale bar = 0.005 mm.</p

Apoptosis marker TUNEL is increased by rLOX-PP treatment.

<p>(A) TUNEL staining of tumors treated i.t. with rLOX-PP, or tumors injected with negative control PBS; (B) tumors treated with surgically implanted empty alginate beads or rLOX-PP alginate beads. Black arrows mark some positive-stained cells; the white arrows point to an inset containing an enlarged image of a stained cell. Scale bar = 0.005 mm for 400× and 0.002 mm for 1000×; (C) the corresponding quantifications (***, p<0.001; n = 3). Arrows mark some positive-stained cells.</p